Optimized binuclease fusions and methods

ABSTRACT

The invention provides for optimized binuclease fusion proteins with increased pharmacokinetic properties. The optimized binuclease fusion proteins of the invention two or more nuclease domains (e.g., RNase and DNase domain) operably coupled to an Fc domain. The invention also provides methods of treating or preventing a condition associated with an abnormal immune response.

RELATED APPLICATIONS

This application claims priority to U.S. provisional Application No.62/357,756 filed Jul. 1, 2016. The contents of the aforementionedapplication are hereby incorporated by reference.

BACKGROUND

Accumulation of (ribo)nucleoprotein particles from dead and dying cellsis known to induce an inflammatory cascade in patients with systemiclupus erythematosus (SLE) by at least two mechanisms: (i) Deposition orin situ formation of chromatin/anti-chromatin complexes causes nephritisand leads to loss of renal function; and (ii) nucleic acids complexedwith autoantibodies activate innate immunity through toll-like receptor(TLR) 7, 8, and 9 as well as TLR-independent pathway(s). Release ofnucleoproteins can serve as a potent antigen for autoantibodies in SLE,providing amplification of B cell and DC activation throughco-engagement of antigen receptors and TLRs. Thus, there exists a needfor a means to remove the nucleic acid bound to autoantibody antigensand/or attenuate immune stimulation, immune amplification, and immunecomplex mediated disease in subjects in need thereof, for example, withlong-acting nuclease molecules that attack circulating immune complexesby digesting nucleic acids contained therein.

SUMMARY OF THE INVENTION

The invention relates, in part, to optimized binuclease fusion proteinswhich are tandem binuclease fusion proteins or heterodimeric binucleasefusion proteins which are capable of binding multiple substrates withhigh nuclease activity. In some aspects, the tandem binuclease fusionproteins comprise one or more DNase1 and one or more RNase1 domainsoperably linked in tandem to one or more Fc domains. In some aspects,the heterodimeric binuclease fusion proteins comprise a single DNase1domain and a single RNase1 domain operably linked to one or more Fcdomains, such that the DNase1 and RNase1 domains are positioned ateither the N- or C-terminus of the Fc domain. In some aspects, theoptimized binuclease fusion proteins alleviate the problem of expressingdual-functional nuclease-Fc chains and mitigate potential sterichindrance of one or more nuclease domains. In some aspects, theheterodimeric binuclease fusion proteins comprise one or more mutationsin the Fc domain(s) to maximize formation of heterodimers.

In some embodiments the optimized binuclease fusion proteins are tandembinuclease fusion proteins comprising a first nuclease domain, a secondnuclease domain and an Fc region, wherein the first nuclease domain isDNase1 and the second nuclease domain is RNase1, wherein the DNase1 isoperably linked with or without a linker in tandem from N- to C-terminusto the RNase1, and the RNase1 is operably linked to the N- or C-terminusof an Fc region. The tandem binuclease fusion protein exhibits enhancedpharmacokinetic activity relative to the either first or second nucleasedomain alone. Such tandem binuclease fusion proteins exhibit altered,e.g., improved, serum half-life relative to either the first or secondnuclease domain alone.

In some aspects the optimized binuclease fusion proteins areheterodimeric binuclease fusion proteins comprising a first nucleasedomain, a second nuclease domain and an Fc region, wherein the firstnuclease domain is DNase1 and the second nuclease domain is RNase1,wherein the DNase1 is operably linked with or without a linker to the N-or C-terminus of an Fc region, and the RNase1 is operably linked with orwithout a linker to the N- or C-terminus of an Fc region, therebyforming a heterodimer. The heterodimeric binuclease fusion proteinexhibits enhanced pharmacokinetic activity relative to the either firstor second nuclease domain alone. Such heterodimeric binuclease fusionproteins exhibit altered, e.g., improved, serum half-life relative toeither the first or second nuclease domain alone.

In some aspect the optimized binuclease fusion proteins are thoserepresented in FIG. 1.

In some aspects, the invention provides an optimized binuclease fusionprotein comprising human DNase1, human RNase1, and a mutant human IgG1Fc, wherein the human DNase1 is operably linked via a linker (e.g., agly-ser linker) to human RNase1, from N-terminus to C terminus, andwherein the human RNase1 is operably linked via a linker to the mutanthuman IgG1 Fc domain wherein the mutant human IgG1 has a mutant hingeregion (e.g., a cysteine substitution, such as with serine, e.g., SCC),and one or more CH2 mutations to reduce Fcγ receptor binding (e.g.,P238S, P331S or both P238S and P331S, numbering according to EU index).In one embodiment, the optimized binuclease fusion protein compriseshuman DNase1 operably linked via a peptide linker (e.g., a gly-serlinker) to human RNase1 (N-terminus-DNase1-linker-RNase1-C terminus) andthe human RNase1 is operably linked via a peptide linker (e.g., agly-ser linker) to a mutant human IgG1 Fc domain having a mutant hingeregion, SCC hinge, and P238S and P331S mutations. In yet anotherembodiment, the Fc domain further includes a mutation at a site ofN-linked glycosylation, such as a substitution at N297 (numbering byKabat).

In some embodiments, the optimized binuclease fusion protein furtherincludes a first linker domain, and the first nuclease domain isoperably coupled to the second nuclease domain, via the first linkerdomain.

In some embodiments, the optimized binuclease fusion protein furtherincludes a second linker domain, and the second nuclease domain isoperably coupled to Fc domain, via the second linker domain.

In some embodiments, the RNase domain is a wild-type RNase, such aswild-type human RNase1. In other embodiments, the RNase domain is amutant RNase, such as an aglycosylated, underglycosylated, ordeglycosylated RNase 1, such as human RNase1 N34S/N76S/N88S (SEQ ID NO:28). In some embodiments, the RNase containing optimized binucleasefusion protein degrades circulating RNA and RNA in immune complexes, orinhibits interferon-alpha production, or both. In yet other embodiments,the activity of the RNase is not less than about 10-fold less, such as9-fold less, 8-fold less, 7-fold less, 6-fold less, 5-fold less, 4-foldless, 3-fold less, or 2-fold less than the activity of a control RNasemolecule. In yet other embodiments, the activity of the RNase is aboutequal to the activity of a control RNase molecule.

In some embodiments, the DNase domain is wild type DNase, such as wildtype, human DNase1. In other embodiments, the DNase domain is a mutantDNase domain, such as mutant, human DNase1 A114F (SEQ ID NO: 21) or anaglycosylated, underglycosylated, or deglycosylated human DNase, such asmutant, human DNase1 N18S/N106S/A114F (SEQ ID NO: 24). In someembodiments, the DNase containing optimized binuclease fusion proteindegrades circulating DNA and DNA in immune complexes, or inhibitsinterferon-alpha production, or both. In yet other embodiments, theactivity of the DNase is not less than about 10-fold less, such as9-fold less, 8-fold less, 7-fold less, 6-fold less, 5-fold less, 4-foldless, 3-fold less, or 2-fold less than the activity of a control DNasemolecule. In yet other embodiments, the activity of the DNase is aboutequal to the activity of a control DNase molecule.

In some embodiments, the optimized binuclease fusion protein has agly-ser linker separating the first and second nuclease domains, and/orthe second nuclease domain from the Fc domain.

In some embodiments, the optimized binuclease fusion protein has anincreased serum half-life and/or activity relative to a molecule thatdoes not contain the Fc domain.

In some aspects, the optimized binuclease fusion protein may include themutant, human DNase1 A114F domain set forth in SEQ ID NO: 21. In anotherembodiment, the optimized binuclease fusion protein may include themutant, human DNase1 N18S/N106S/A114F domain set forth in SEQ ID NO: 24.In some embodiments, the DNase domain is mutant human DNase1E13R/N74K/A114F/T205K (SEQ ID NO: 25). In other embodiments, the DNasedomain is mutant human DNase1 E13R/N74K/A114F/T205K/N18S/N106S (SEQ IDNO: 26).

In some embodiments, DNase1 and RNase1 domains are aglycosylated,underglycosylated, or deglycosylated. In some embodiments, the DNasedomain is a mutant DNase domain, such as mutant, human DNase1 and anaglycosylated, underglycosylated, or deglycosylated DNase domain, suchas an aglycosylated, underglycosylated, or deglycosylated human DNase1.In one embodiment, the human DNase1 includes an alteration (e.g., asubstitution) at one or more sites of N-linked glycosylation, such asN18 and N106 and at least one additional mutation selected from A114,E13, N74, T205, and combinations thereof. In another embodiment, thehuman DNase1 includes an alteration (e.g., a substitution) at N18, N106,or both N18 and N106 and an additional alertation (e.g., a substitution)at A114, E13, N74, T205, and combinations thereof. In yet anotherembodiment, the human DNase1 includes an alteration at N18, N106, A114,E13, N74 and T205, such as a substitution, e.g.,N18S/N106S/A114F/E13R/N74K/T205K (SEQ ID NO: 26). In another embodiment,the optimized binuclease fusion protein with altered glycosylationincludes the human, wild-type RNase1 domain set forth in SEQ ID NO: 27In another embodiment, the optimized binuclease fusion protein withaltered glycosylation includes the human, mutant RNase1 N34S/N76S/N88Sdomain set forth in SEQ ID NO: 28.

In some aspects, the invention provides an optimized binuclease fusionprotein comprising the polypeptides having an amino acid sequence setforth in SEQ ID NOs: 1-17. In other aspects, the optimized binucleasefusion protein have an amino acid sequence at least 90% identical or atleast 95% identical to an amino acid sequence set forth in SEQ ID NOs:1-17.

In some aspects, the optimized binuclease fusion protein comprises apolypeptide comprising a first nuclease domain, a second nuclease domainand an Fc domain, wherein the first nuclease domain is DNase1 and thesecond nuclease domain is RNase1, wherein the DNase1 is operably linkedwith or without a linker in tandem from N- to C-terminus to the RNase1,and the RNase1 is operably linked with or without a linker to the Fcregion. In some aspects, the RNase 1 is operably linked to theN-terminus of the Fc domain without a linker. In some aspects, the RNase1 is operably linked to the C-terminus of the Fc domain without alinker. In some aspects, the DNase1 is operably linked to the RNase1 viaa linker. In some aspects, the polypeptide comprise an amino acidsequence set forth in SEQ ID NO: 1 or SEQ ID NO:2, or a tandembinuclease fusion protein comprising an amino acid sequence at least 90%identical to the amino acid sequence set forth in SEQ ID NO:1 or SEQ IDNO:2. In some aspects, the tandem binuclease fusion protein is ahomodimer comprising any of the foregoing polypeptides.

In some aspects, the optimized binuclease fusion protein is aheterodimer comprising a first nuclease domain, a second nucleasedomain, a first Fc domain and a second Fc domain, wherein the firstnuclease domain is DNase1 and the second nuclease domain is RNase1,wherein the DNase1 is operably linked with or without a linker to the N-or C-terminus of the first Fc domain, and the RNase1 is operably linkedwith or without a linker to the N- or C-terminus of the second Fcdomain. In some aspects of the foregoing heterodimer, the DNase 1 isoperably linked without a linker to the N-terminus of the first Fcdomain and the RNase1 is operably linked without a linker to theN-terminus of the second Fc domain.

In some aspects, the DNase 1 is operably linked with a linker to theN-terminus of the first Fc domain and the RNase1 is operably linked witha linker to the N-terminus of the second Fc domain. In some aspects, theDNase 1 is operably linked with a linker to the N-terminus of the firstFc domain and the RNase1 is operably linked without a linker to theC-terminus of the second Fc domain. In some aspects, the DNase 1 isoperably linked without a linker to the N-terminus of the first Fcdomain and the RNase1 is operably linked without a linker to theC-terminus of the second Fc domain. In some aspects the DNase 1 isoperably linked with a linker to the N-terminus of the first Fc domainand the RNase1 is operably linked with a linker to the C-terminus of thesecond Fc domain. In some aspects, the DNase 1 is operably linked with alinker to the C-terminus of the first Fc domain and the RNase1 isoperably linked with a linker to the C-terminus of the second Fc domain.In some aspects, the DNase 1 is operably linked without a linker to theC-terminus of the first Fc domain and the RNase1 is operably linkedwithout a linker to the C-terminus of the second Fc domain. In someaspects, the DNase 1 is operably linked with a linker to the C-terminusof the first Fc domain and the RNase1 is operably linked without alinker to the N-terminus of the second Fc domain. In some aspects, theDNase 1 is operably linked with a linker to the C-terminus of the firstFc domain and the RNase1 is operably linked with a linker to theN-terminus of the second Fc domain.

In some aspects, the optimized binuclease fusion protein is aheterodimer comprising a first and second polypeptide sequence selectedfrom the group consisting of:

(i) a first polypeptide comprising an amino acid sequence set forth inSEQ ID NO: 3, or a polypeptide comprising an amino acid sequence atleast 90% identical to the amino acid sequence set forth in SEQ ID NO:3;and a second polypeptide comprising an amino acid sequence set forth inSEQ ID NO: 4, or a polypeptide comprising an amino acid sequence atleast 90% identical to the amino acid sequence set forth in SEQ ID NO:4, or

(ii) a first polypeptide comprising an amino acid sequence set forth inSEQ ID NO: 7, or a polypeptide comprising an amino acid sequence atleast 90% identical to the amino acid sequence set forth in SEQ ID NO:7;and a second polypeptide comprising an amino acid sequence set forth inSEQ ID NO: 8, or a polypeptide comprising an amino acid sequence atleast 90% identical to the amino acid sequence set forth in SEQ ID NO:8,or

(iii) a first polypeptide comprising an amino acid sequence set forth inSEQ ID NO:9, or a polypeptide comprising an amino acid sequence at least90% identical to the amino acid sequence set forth in SEQ ID NO:9; and asecond polypeptide comprising an amino acid sequence set forth in SEQ IDNO: 10, or a polypeptide comprising an amino acid sequence at least 90%identical to the amino acid sequence set forth in SEQ ID NO:10, or

(iv) a first polypeptide comprising an amino acid sequence set forth inSEQ ID NO: 11, or a polypeptide comprising an amino acid sequence atleast 90% identical to the amino acid sequence set forth in SEQ ID NO:11; and a second polypeptide comprising an amino acid sequence set forthin SEQ ID NO: 12, or a polypeptide comprising an amino acid sequence atleast 90% identical to the amino acid sequence set forth in SEQ IDNO:12, or

(v) a first polypeptide comprising an amino acid sequence set forth inSEQ ID NO: 15, or a polypeptide comprising an amino acid sequence atleast 90% identical to the amino acid sequence set forth in SEQ ID NO:15; and a second polypeptide comprising an amino acid sequence set forthin SEQ ID NO: 16, or a polypeptide comprising an amino acid sequence atleast 90% identical to the amino acid sequence set forth in SEQ IDNO:16.

Other aspects relate to a heterodimer comprising a first nucleasedomain, a second nuclease domain and a first Fc domain and a second Fcdomain, wherein the first nuclease domain is DNase1 and the secondnuclease domain is RNase1, wherein

-   -   (i) the DNase1 is operably linked with or without a linker to        the N-terminus of the first Fc domain, and the RNase1 is        operably linked with or without a linker to the C-terminus of        the first Fc domain, or    -   (ii) the RNase1 is operably linked with or without a linker to        the N-terminus of the first Fc domain, and the DNase1 is        operably linked with or without a linker to the C-terminus of        the first Fc domain.

In some aspects of the foregoing heterodimer, the DNase 1 is operablylinked without a linker to the N-terminus of the first Fc domain and theRNase1 is operably linked with a linker to the C-terminus of the firstFc domain. In some aspects, the DNase 1 is operably linked with a linkerto the N-terminus of the first Fc domain and the RNase1 is operablylinked with a without a linker to the C-terminus of the first Fc domain.In some aspects, the RNase 1 is operably linked without a linker to theN-terminus of the first Fc domain and the DNase 1 is operably linkedwith a linker to the C-terminus of the first Fc domain. In some aspects,the RNase 1 is operably linked with a linker to the N-terminus of thefirst Fc domain and the DNase 1 is operably linked with a linker to theC-terminus of the first Fc domain.

In some aspects, a heterodimer comprises a first and second polypeptidesequence selected from the group consisting of:

(i) a first polypeptide comprising an amino acid sequence set forth inSEQ ID NO:5, or a polypeptide comprising an amino acid sequence at least90% identical to the amino acid sequence set forth in SEQ ID NO:5; and asecond polypeptide comprising an amino acid sequence set forth in SEQ IDNO:6, or a polypeptide comprising an amino acid sequence at least 90%identical to the amino acid sequence set forth in SEQ ID NO:6, or

(ii) a first polypeptide comprising an amino acid sequence set forth inSEQ ID NO: 13, or a polypeptide comprising an amino acid sequence atleast 90% identical to the amino acid sequence set forth in SEQ ID NO:13; and a second polypeptide comprising an amino acid sequence set forthin SEQ ID NO: 14, or a polypeptide comprising an amino acid sequence atleast 90% identical to the amino acid sequence set forth in SEQ IDNO:14.

In some aspects, any of the foregoing heterodimers comprise one or moreCH3 mutations in the Fc domains to preferentially form heterodimers. Insome aspects, the heterodimer comprises a first Fc domain comprising CH3mutations T350V, L351Y, F405A, and Y407V, and a second Fc domaincomprising CH3 mutations T350V, T366L, K392L, T394W, numbering accordingto the EU index.

Other aspects of the disclosure relate to compositions comprising any ofthe foregoing heterodimer and a pharmaceutically acceptable carrier.Nucleic acid molecules encoding the foregoing heterodimers, recombinantexpression vectors and host cell transformed with the recombinantexpression vectors, as well as methods of making the foregoingheterodimers are also disclosed.

Also disclosed herein is a method of making a tandem optimizedbinuclease fusion protein disclosed herein involving providing a hostcell comprising a nucleic acid sequence that encodes the optimizedbinuclease fusion protein; and maintaining the host cell underconditions in which the optimized binuclease fusion protein isexpressed.

Also disclosed herein is a method for treating or preventing a conditionassociated with an abnormal immune response by administering to apatient in need thereof an effective amount of a optimized binucleasefusion protein disclosed herein. In some embodiments, the condition isan autoimmune disease. In some embodiments, the autoimmune disease isselected from the group consisting of insulin-dependent diabetesmellitus, multiple sclerosis, experimental autoimmune encephalomyelitis,rheumatoid arthritis, experimental autoimmune arthritis, myastheniagravis, thyroiditis, an experimental form of uveoretinitis, Hashimoto'sthyroiditis, primary myxoedema, thyrotoxicosis, pernicious anaemia,autoimmune atrophic gastritis, IgG4 related disease, Addison's disease,premature menopause, male infertility, juvenile diabetes, Goodpasture'ssyndrome, pemphigus vulgaris, pemphigoid, sympathetic ophthalmia,phacogenic uveitis, autoimmune haemolytic anaemia, idiopathicleucopenia, primary biliary cirrhosis, active chronic hepatitis Hbs-ve,cryptogenic cirrhosis, ulcerative colitis, Sjogren's syndrome,scleroderma, Wegener's granulomatosis, polymyositis, dermatomyositis,discoid LE, systemic lupus erythematosus (SLE), and connective tissuedisease. In some embodiments, the autoimmune disease is SLE or Sjogren'ssyndrome.

Also disclosed herein is a method of treating SLE or Sjogren's syndromecomprising administering to a subject a optimized binuclease fusionprotein containing composition in an amount effective to degrade immunecomplexes containing RNA, DNA or both RNA and DNA. In some aspects, thecomposition includes a pharmaceutically acceptable carrier and aoptimized binuclease fusion protein as described herein. In otheraspects, the composition includes a optimized binuclease fusion proteinhaving an amino acid sequence set forth in SEQ ID NO: 1.

In another aspect, the invention relates to optimized binuclease fusionproteins for use in treating diseases characterized by defectiveclearance or processing of apoptotic cells and cell debris, such as SLE.In some embodiments, the optimized binuclease fusion protein comprisesamino acid sequences set forth in SEQ ID NOs: 4 and 5.

In another aspect, the invention relates to the use of the optimizedbinuclease fusion proteins for manufacturing a medicament for treatingdiseases characterized by defective clearance or processing of apoptoticcells and cell debris, such as SLE. In some embodiments, the optimizedbinuclease fusion protein comprises amino acid sequences set forth inSEQ ID NOs: 5 and 6.

In another aspect, the invention relates to the use of the optimizedbinuclease fusion proteins for manufacturing a medicament for treatingdiseases characterized by defective clearance or processing of apoptoticcells and cell debris, such as SLE. In some embodiments, the optimizedbinuclease fusion protein comprises amino acid sequences set forth inSEQ ID NOs: 7 and 8.

In another aspect, the invention relates to the use of the optimizedbinuclease fusion proteins for manufacturing a medicament for treatingdiseases characterized by defective clearance or processing of apoptoticcells and cell debris, such as SLE. In some embodiments, the optimizedbinuclease fusion protein comprises amino acid sequences set forth inSEQ ID NOs: 13 and 14.

In another aspect, the invention relates to the use of the optimizedbinuclease fusion proteins for manufacturing a medicament for treatingdiseases characterized by defective clearance or processing of apoptoticcells and cell debris, such as SLE. In some embodiments, the optimizedbinuclease fusion protein comprises amino acid sequences set forth inSEQ ID NOs: 15 and 16.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects, and advantages of the presentinvention will become better understood with regard to the followingdescription, and accompanying drawing, where:

FIG. 1 is a depiction of exemplary optimized binuclease fusion proteins.

FIG. 2 is a graph showing RNase activity as measured by OD₂₆₀.

FIG. 3 shows DNase activity as measured by OD₆₂₀ (left) and IC₅₀(right).

DETAILED DESCRIPTION

Systemic lupus erythematosus (SLE) is a multisystem autoimmune diseasecharacterized by the presence of high titer autoantibodies directedagainst self nucleoproteins. There is strong evidence that defectiveclearance or processing of dead and dying cells in SLE leads to disease,predominantly through accumulation of ribo- and deoxy-ribonucleoproteins(abbreviated nucleoproteins). The nucleoproteins cause damage throughthree mechanisms: i) activation of the innate immune system to produceinflammatory cytokines; ii) serve as antigens to generate circulatingimmune complexes; and iii) serve as antigens to generate in situ complexformation at local sites such as the kidney.

The present invention provides methods for treating diseasescharacterized by defective clearance or processing of apoptotic cellsand cell debris, such as SLE and Sjogren's syndrome, by administering aneffective amount of a long-acting nuclease activity to degradeextracellular RNA and DNA containing immune complexes. Such treatmentcan inhibit production of Type I interferons (IFNs) which are prominentcytokines in SLE and are strongly correlated with disease activity andnephritis.

The present invention relates, in part, to the provision of suchlong-acting nucleases. In particular, the invention relates to anoptimized binuclease fusion protein, such as a tandem binuclease fusionprotein comprising a first nuclease domain, a second nuclease domain andan Fc region, wherein the first nuclease domain is DNase1 and the secondnuclease domain is RNase1, wherein the DNase1 is operably linked with orwithout a linker in tandem from N- to C-terminus to the RNase1, and theRNase1 is operably linked to the N- or C-terminus of an Fc region.

In other embodiments, the invention relates to an optimized binucleasefusion protein, such as a heterodimeric binuclease fusion proteincomprising a first nuclease domain, a second nuclease domain and an Fcregion, wherein the first nuclease domain is DNase1 and the secondnuclease domain is RNase1, wherein the DNase1 is operably linked with orwithout a linker to the N- or C-terminus of an Fc region, and the RNase1is operably linked with or without a linker to the N- or C-terminus ofan Fc region, thereby forming a heterodimer.

In some aspects, the optimized binuclease fusion protein exhibitsenhanced pharmacokinetic activity relative to the either first or secondnuclease domain alone. Such optimized binuclease fusion proteins exhibitaltered, e.g., improved, serum half-life relative to either the first orsecond nuclease domain alone.

In some aspects, the invention provides a optimized binuclease fusionprotein comprising human DNase1, human RNase1, and a mutant human IgG1Fc, wherein the human DNase1 is operably linked via a linker (e.g., agly-ser linker) to human RNase1, from N-terminus to C terminus, andwherein the human RNase1 is operably linked via a linker to the mutanthuman IgG1 Fc domain wherein the mutant human IgG1 has a mutant hingeregion (e.g., a cysteine substitution, such as with serine, e.g., SCC),and one or more CH2 mutations to reduce Fcγ receptor binding (e.g.,P238S, P331S or both P238S and P331S, numbering according to EU index).In one embodiment, the optimized binuclease fusion protein compriseshuman DNase1 operably linked via a peptide linker (e.g., a gly-serlinker) to human RNase1 (N-terminus-DNase1-linker-RNase1-C terminus) andthe human RNase1 is operably linked via a peptide linker (e.g., agly-ser linker) to a mutant human IgG1 Fc domain having a mutant hingeregion, SCC hinge, and P238S and P331S mutations.

Accordingly, in one embodiment, a subject with a disease characterizedby defective clearance or processing of apoptotic cells and cell debrisis treated by administering a optimized binuclease fusion protein, whichincludes both DNase1 and RNase1, such that the optimized binucleasefusion protein has increased bioavailability and/or serum half-liferelative to the non-conjugated nuclease domains.

Definitions

Terms used in the claims and specification are defined as set forthbelow unless otherwise specified.

“Amino acid” refers to naturally occurring and synthetic amino acids, aswell as amino acid analogs and amino acid mimetics that function in amanner similar to the naturally occurring amino acids. Naturallyoccurring amino acids are those encoded by the genetic code, as well asthose amino acids that are later modified, e.g., hydroxyproline,γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers tocompounds that have the same basic chemical structure as a naturallyoccurring amino acid, i.e., an a carbon that is bound to a hydrogen, acarboxyl group, an amino group, and an R group, e.g., homoserine,norleucine, methionine sulfoxide, methionine methyl sulfonium. Suchanalogs have modified R groups (e.g., norleucine) or modified peptidebackbones, but retain the same basic chemical structure as a naturallyoccurring amino acid. Amino acid mimetics refer to chemical compoundsthat have a structure that is different from the general chemicalstructure of an amino acid, but that function in a manner similar to anaturally occurring amino acid.

Amino acids can be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,can be referred to by their commonly accepted single-letter codes.

An “amino acid substitution” refers to the replacement of at least oneexisting amino acid residue in a predetermined amino acid sequence (anamino acid sequence of a starting polypeptide) with a second, different“replacement” amino acid residue. An “amino acid insertion” refers tothe incorporation of at least one additional amino acid into apredetermined amino acid sequence. While the insertion will usuallyconsist of the insertion of one or two amino acid residues, larger“peptide insertions” can be made, e.g. insertion of about three to aboutfive or even up to about ten, fifteen, or twenty amino acid residues.The inserted residue(s) may be naturally occurring or non-naturallyoccurring as disclosed above. An “amino acid deletion” refers to theremoval of at least one amino acid residue from a predetermined aminoacid sequence.

“Polypeptide,” “peptide,” and “protein” are used interchangeably hereinto refer to a polymer of amino acid residues. The terms apply to aminoacid polymers in which one or more amino acid residue is an artificialchemical mimetic of a corresponding naturally occurring amino acid, aswell as to naturally occurring amino acid polymers and non-naturallyoccurring amino acid polymer.

“Nucleic acid” refers to deoxyribonucleotides or ribonucleotides andpolymers thereof in either single- or double-stranded form. Unlessspecifically limited, the term encompasses nucleic acids containingknown analogues of natural nucleotides that have similar bindingproperties as the reference nucleic acid and are metabolized in a mannersimilar to naturally occurring nucleotides. Unless otherwise indicated,a particular nucleic acid sequence also implicitly encompassesconservatively modified variants thereof (e.g., degenerate codonsubstitutions) and complementary sequences, as well as the sequenceexplicitly indicated. Specifically, degenerate codon substitutions canbe achieved by generating sequences in which the third position of oneor more selected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues (Batzer et al., Nucleic Acid Res 1991; 19:5081;Ohtsuka et al., JBC 1985; 260:2605-8); Rossolini et al., Mol Cell Probes1994; 8:91-8). For arginine and leucine, modifications at the secondbase can also be conservative. The term nucleic acid is usedinterchangeably with gene, cDNA, and mRNA encoded by a gene.

Polynucleotides of the present invention can be composed of anypolyribonucleotide or polydeoxribonucleotide, which can be unmodifiedRNA or DNA or modified RNA or DNA. For example, polynucleotides can becomposed of single- and double-stranded DNA, DNA that is a mixture ofsingle- and double-stranded regions, single- and double-stranded RNA,and RNA that is mixture of single- and double-stranded regions, hybridmolecules comprising DNA and RNA that can be single-stranded or, moretypically, double-stranded or a mixture of single- and double-strandedregions. In addition, the polynucleotide can be composed oftriple-stranded regions comprising RNA or DNA or both RNA and DNA. Apolynucleotide can also contain one or more modified bases or DNA or RNAbackbones modified for stability or for other reasons. “Modified” basesinclude, for example, tritylated bases and unusual bases such asinosine. A variety of modifications can be made to DNA and RNA; thus,“polynucleotide” embraces chemically, enzymatically, or metabolicallymodified forms.

As used herein, the term “operably linked” or “operably coupled” refersto a juxtaposition wherein the components described are in arelationship permitting them to function in their intended manner.

As used herein, the term “glycosylation” or “glycosylated” refers to aprocess or result of adding sugar moieties to a molecule (e.g., anoptimized binuclease fusion protein).

As used herein, the term “altered glycosylation” refers to a moleculethat is aglycosylated, deglycosylated, or underglycosylated.

As used herein, “glycosylation site(s)” refers to both sites thatpotentially could accept a carbohydrate moiety, as well as sites withinthe protein on which a carbohydrate moiety has actually been attachedand includes any amino acid sequence that could act as an acceptor foran oligosaccharide and/or carbohydrate.

As used herein, the term “aglycosylation” or “aglycosylated” refers tothe production of a molecule (e.g., an optimized binuclease fusionprotein) in an unglycosylated form (e.g., by engineering an optimizedbinuclease fusion protein to lack amino acid residues that serve asacceptors of glycosylation). Alternatively, the optimized binucleasefusion protein can be expressed in, e.g., E. coli, to produce anaglycosylated optimized binuclease fusion protein.

As used herein, the term “deglycosylation” or “deglycosylated” refers tothe process or result of enzymatic removal of sugar moieties on amolecule.

As used herein, the term “underglycosylation” or “underglycosylated”refers to a molecule in which one or more carbohydrate structures thatwould normally be present if produced in a mammalian cell has beenomitted, removed, modified, or masked.

As used herein, the term “Fc region” and “Fc domain” is the portion of anative immunoglobulin formed by the respective Fc domains (or Fcmoieties) of its two heavy chains without the variable regions whichbind antigen. In some embodiments, an Fc domain begins in the hingeregion just upstream of the papain cleavage site and ending at theC-terminus of the antibody. Accordingly, a complete Fc domain comprisesat least a hinge domain, a CH2 domain, and a CH3 domain. In certainembodiments, an Fc domain comprises at least one of: a hinge (e.g.,upper, middle, and/or lower hinge region) domain, a CH2 domain, a CH3domain, a CH4 domain, or a variant, portion, or fragment thereof. Inother embodiments, an Fc domain comprises a complete Fc domain (i.e., ahinge domain, a CH2 domain, and a CH3 domain). In one embodiment, an Fcdomain comprises a hinge domain (or portion thereof) fused to a CH3domain (or portion thereof). In another embodiment, an Fc domaincomprises a CH2 domain (or portion thereof) fused to a CH3 domain (orportion thereof). In another embodiment, an Fc domain consists of a CH3domain or portion thereof. In another embodiment, an Fc domain consistsof a hinge domain (or portion thereof) and a CH3 domain (or portionthereof). In another embodiment, an Fc domain consists of a CH2 domain(or portion thereof) and a CH3 domain. In another embodiment, an Fcdomain consists of a hinge domain (or portion thereof) and a CH2 domain(or portion thereof). In one embodiment, an Fc domain lacks at least aportion of a CH2 domain (e.g., all or part of a CH2 domain). In oneembodiment, an Fc domain of the invention comprises at least the portionof an Fc molecule known in the art to be required for FcRn binding. Inone embodiment, an Fc domain of the invention comprises at least theportion of an Fc molecule known in the art to be required for Protein Abinding. In one embodiment, an Fc domain of the invention comprises atleast the portion of an Fc molecule known in the art to be required forprotein G binding. An Fc domain herein generally refers to a polypeptidecomprising all or part of the Fc domain of an immunoglobulinheavy-chain. This includes, but is not limited to, polypeptidescomprising the entire CHI, hinge, CH2, and/or CH3 domains as well asfragments of such peptides comprising only, e.g., the hinge, CH2, andCH3 domain. The Fc domain may be derived from an immunoglobulin of anyspecies and/or any subtype, including, but not limited to, a human IgG1,IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibody. The Fc domainencompasses native Fc and Fc variant molecules. As with Fc variants andnative Fc's, the term Fc domain includes molecules in monomeric ormultimeric form, whether digested from whole antibody or produced byother means.

As set forth herein, it will be understood by one of ordinary skill inthe art that any Fc domain may be modified such that it varies in aminoacid sequence from the native Fc domain of a naturally occurringimmunoglobulin molecule.

The Fc domains of an optimized binuclease fusion protein of thedisclosure may be derived from different immunoglobulin molecules. Forexample, an Fc domain of an optimized binuclease fusion protein maycomprise a CH2 and/or CH3 domain derived from an IgG1 molecule and ahinge region derived from an IgG3 molecule. In another example, an Fcdomain can comprise a chimeric hinge region derived, in part, from anIgG1 molecule and, in part, from an IgG3 molecule. In another example,an Fc domain can comprise a chimeric hinge derived, in part, from anIgG1 molecule and, in part, from an IgG4 molecule. The wild type humanIgG1 Fc domain has the amino acid sequence set forth in SEQ ID NO: 45.

As used herein, the term “serum half-life” refers to the time requiredfor the in vivo serum optimized binuclease fusion protein concentrationto decline by 50%. The shorter the serum half-life of the optimizedbinuclease fusion protein, the shorter time it will have to exert atherapeutic effect.

As used herein, the term “optimized binuclease fusion protein” refers topolypeptides that comprise at least two nuclease domains operablylinked, with or without a linker, to an Fc domain, or a variant orfragment thereof, and nucleic acids encoding such polypeptides. In someembodiments, an optimized binuclease fusion protein is a tandembinuclease fusion protein, e.g., a one or more DNase 1 domains and oneor more RNase 1 domains linked in tandem to either the N- or C-terminusof one or more Fc domain. In some embodiments, an optimized binucleasefusion protein is a heterodimeric binuclease fusion protein.

As used herein, the term “tandem binuclease fusion protein” refers to apolypeptide that comprises at least two nuclease domains linked intandem (from N- to C-terminus) and an Fc domain, or a variant orfragment thereof, and nucleic acids encoding such polypeptides. Forexample, in one embodiment, a tandem binuclease fusion protein is apolypeptide comprising at least one DNase1 domain and at least oneRNase1 domain operably linked in tandem to at least one Fc domain. Asanother example, a tandem binuclease fusion protein includes from N- toC-terminus a DNase1 domain, a first linker, an RNase1 domain, a secondlinker, and an Fc domain, or a variant or fragment thereof.

As used herein, the term “heterodimeric binuclease fusion protein”refers to a heterodimer comprising a first and a second polypeptide,which together comprise at least two nuclease domains and two Fcdomains, variants or fragment thereof, and nucleic acids encoding suchpolypeptides. In some embodiments, a heterodimeric binuclease fusionprotein is a heterodimer comprising at least one DNase1 domain and atleast one RNase1 domain operably linked to at least one Fc domain,wherein the DNase 1 domain is operably linked with or without a linkerto the N- or C-terminus of a first Fc domain and an RNase1 domain isoperably linked with or without a linker the N- or C-terminus of a same(first Fc domain) or a different Fc domain (second Fc domain), such thatthe DNase 1 domain and the RNase 1 domain are located on opposite ends(N- or C-terminus) of either the same (first Fc domain) or different Fcdomain (second Fc domain). In some embodiments, the heterodimercomprises a DNase 1 domain operably linked with or without a linker tothe N- or C-terminus of a first Fc domain, and a RNase 1 operably linkedwith or without a linker to the N- or C-terminus of the second Fcdomain, such that the DNase 1 and RNase 1 domains are located at thesame end (N- or C-terminus) of the heterodimer in tandem. In someembodiments, the heterodimer comprises a DNase 1 domain operably linkedwith or without a linker to the N-terminus of a first Fc domain, and aRNase 1 operably linked with or without a linker to the C-terminus ofthe first Fc domain. In some embodiments, the RNase1 is operably linkedwith or without a linker to the N-terminus of the first Fc domain, andthe DNase1 is operably linked with or without a linker to the C-terminusof the first Fc domain.

As used herein, the term “variant” refers to a polypeptide derived froma wild-type nuclease or Fc domain and differs from the wild-type by oneor more alteration(s), i.e., a substitution, insertion, and/or deletion,at one or more positions. A substitution means a replacement of an aminoacid occupying a position with a different amino acid. A deletion meansremoval of an amino acid occupying a position. An insertion means adding1 or more, such as 1-3 amino acids, immediately adjacent to an aminoacid occupying a position. Variant polypeptides necessarily have lessthan 100% sequence identity or similarity with the wild-typepolypeptide. In some embodiments, the variant polypeptide will have anamino acid sequence from about 75% to less than 100% amino acid sequenceidentity or similarity with the amino acid sequence of wild-typepolypeptide, or from about 80% to less than 100%, or from about 85% toless than 100%, or from about 90% to less than 100% (e.g., 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% o, 99%) or from about 95% to less than100%, e.g., over the length of the variant polypeptide.

In certain aspects, the optimized binuclease fusion proteins employ oneor more “linker domains,” such as polypeptide linkers. As used herein,the term “linker domain” refers to one or more amino acids which connecttwo or more peptide domains in a linear polypeptide sequence. As usedherein, the term “polypeptide linker” refers to a peptide or polypeptidesequence (e.g., a synthetic peptide or polypeptide sequence) whichconnects two or more polypeptide domains in a linear amino acid sequenceof a protein. For example, polypeptide linkers may be used to operablylink a first and second nuclease domain to each other, or a first orsecond nuclease domain to an Fc domain. Such polypeptide linkers in someembodiments provide flexibility to the polypeptide molecule. In someembodiments the polypeptide linker is used to connect (e.g., geneticallyfuse) a DNase1 to an RNase1 and/or RNase1 to an Fc domain. An optimizedbinuclease fusion protein may include more than one linker domain orpeptide linker. Various peptide linkers are known in the art.

As used herein, the term “gly-ser polypeptide linker” refers to apeptide that consists of glycine and serine residues. An exemplarygly/ser polypeptide linker comprises the amino acid sequence (Gly₄Ser)n.In some embodiments, n is 1 or more, such as 2 or more, 3 or more, 4 ormore, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 ormore (e.g., (Gly₄Ser)10). Another exemplary gly/ser polypeptide linkercomprises the amino acid sequence Ser(Gly₄Ser)n. In some embodiments, nis 1 or more, such as 2 or more, 3 or more, 4 or more, 5 or more, 6 ormore, 7 or more, 8 or more, 9 or more, or 10 or more (e.g.,Ser(Gly₄Ser)10).

As used herein, the terms “coupled,” “linked,” “fused,” or “fusion,” areused interchangeably. These terms refer to the joining together of twomore elements or components or domains, by whatever means includingchemical conjugation or recombinant means. Methods of chemicalconjugation (e.g., using heterobifunctional crosslinking agents) areknown in the art.

A polypeptide or amino acid sequence “derived from” a designatedpolypeptide or protein refers to the origin of the polypeptide.Preferably, the polypeptide or amino acid sequence which is derived froma particular sequence has an amino acid sequence that is essentiallyidentical to that sequence or a portion thereof, wherein the portionconsists of at least 10-20 amino acids, preferably at least 20-30 aminoacids, more preferably at least 30-50 amino acids, or which is otherwiseidentifiable to one of ordinary skill in the art as having its origin inthe sequence. Polypeptides derived from another peptide may have one ormore mutations relative to the starting polypeptide, e.g., one or moreamino acid residues which have been substituted with another amino acidresidue or which has one or more amino acid residue insertions ordeletions.

In one embodiment, there is one amino acid difference between a startingpolypeptide sequence and the sequence derived therefrom. Identity orsimilarity with respect to this sequence is defined herein as thepercentage of amino acid residues in the candidate sequence that areidentical (i.e., same residue) with the starting amino acid residues,after aligning the sequences and introducing gaps, if necessary, toachieve the maximum percent sequence identity.

In one embodiment, a polypeptide of the disclosure consists of, consistsessentially of, or comprises an amino acid sequence as set forth in theSequence Listing or Sequence Table disclosed herein and functionallyactive variants thereof. In an embodiment, a polypeptide includes anamino acid sequence at least 80%, such as at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% identical to an amino acid sequence set forthin the Sequence Listing or Sequence Table disclosed herein. In someembodiments, a polypeptide includes a contiguous amino acid sequence atleast 80%, such as at least 81%, at least 82%, at least 83%, at least84%, at least 85%, at least 86%, at least 87%, at least 88%, at least89%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99% identical to a contiguous amino acid sequence set forth in theSequence Listing or Sequence Table disclosed herein. In someembodiments, a polypeptide includes an amino acid sequence having atleast 10, such as at least 15, at least 20, at least 25, at least 30, atleast 35, at least 40, at least 45, at least 50, at least 55, at least60, at least 65, at least 70, at least 75, at least 80, at least 85, atleast 90, at least 95, at least 100, at least 200, at least 300, atleast 400, or at least 500 (or any integer within these numbers)contiguous amino acids of an amino acid sequence set forth in SequenceListing or Sequence Table disclosed herein.

In some embodiments, the optimized binuclease fusion proteins of thedisclosure are encoded by a nucleotide sequence. Nucleotide sequences ofthe disclosure can be useful for a number of applications, including:cloning, gene therapy, protein expression and purification, mutationintroduction, DNA vaccination of a host in need thereof, antibodygeneration for, e.g., passive immunization, PCR, primer and probegeneration, siRNA design and generation (see, e.g., the DharmaconsiDesign website), and the like. In some embodiments, the nucleotidesequence of the disclosure comprises, consists of, or consistsessentially of, a nucleotide sequence that encodes the amino acidsequence of the optimized binuclease fusion proteins selected from theSequence Table or Sequence Listing. In some embodiments, a nucleotidesequence includes a nucleotide sequence at least 80%, such as at least81%, at least 82%, at least 83%, at least 84%, at least 85%, at least86%, at least 87%, at least 88%, at least 89%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99% identical to anucleotide sequence encoding an amino acid sequence of the SequenceListing or Sequence Table disclosed herein. In some embodiments, anucleotide sequence includes a contiguous nucleotide sequence at least80%, such as at least 81%, at least 82%, at least 83%, at least 84%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to a contiguous nucleotide sequence encoding an amino acidsequence set forth in the Sequence Listing or Sequence Table disclosedherein. In some embodiments, a nucleotide sequence includes a nucleotidesequence having at least 10, such as at least 15, such as at least 20,at least 25, at least 30, at least 35, at least 40, at least 45, atleast 50, at least 55, at least 60, at least 65, at least 70, at least75, at least 80, at least 85, at least 90, at least 95, at least 100, atleast 200, at least 300, at least 400, or at least 500 (or any integerwithin these numbers) contiguous nucleotides of a nucleotide sequenceencoding an amino acid sequence set forth in the Sequence Listing orSequence Table disclosed herein.

It will also be understood by one of ordinary skill in the art that theoptimized binuclease fusion proteins may be altered such that they varyin sequence from the naturally occurring or native sequences from whichtheir components (e.g., nuclease domains, linker domains, and Fcdomains) are derived, while retaining the desirable activity of thenative sequences. For example, nucleotide or amino acid substitutionsleading to conservative substitutions or changes at “non-essential”amino acid residues may be made. An isolated nucleic acid moleculeencoding a non-natural variant can be created by introducing one or morenucleotide substitutions, additions or deletions into the nucleotidesequence of the optimized binuclease fusion protein such that one ormore amino acid substitutions, additions or deletions are introducedinto the encoded protein. Mutations may be introduced by standardtechniques, such as site-directed mutagenesis and PCR-mediatedmutagenesis.

The optimized binuclease fusion proteins may comprise conservative aminoacid substitutions at one or more amino acid residues, e.g., atessential or non-essential amino acid residues. A “conservative aminoacid substitution” is one in which the amino acid residue is replacedwith an amino acid residue having a similar side chain. Families ofamino acid residues having similar side chains have been defined in theart, including basic side chains (e.g., lysine, arginine, histidine),acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polarside chains (e.g., glycine, asparagine, glutamine, serine, threonine,tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine), andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Thus, a nonessential amino acid residue in an optimizedbinuclease fusion protein is preferably replaced with another amino acidresidue from the same side chain family. In another embodiment, a stringof amino acids can be replaced with a structurally similar string thatdiffers in order and/or composition of side chain family members.Alternatively, in another embodiment, mutations may be introducedrandomly along all or part of a coding sequence, such as by saturationmutagenesis, and the resultant mutants can be incorporated into theoptimized binuclease fusion proteins and screened for their ability tobind to the desired target.

The term “ameliorating” refers to any therapeutically beneficial resultin the treatment of a disease state, e.g., an autoimmune disease state(e.g., SLE, Sjogren's syndrome), including prophylaxis, lessening in theseverity or progression, remission, or cure thereof.

The term “in situ” refers to processes that occur in a living cellgrowing separate from a living organism, e.g., growing in tissueculture.

The term “in vivo” refers to processes that occur in a living organism.

The term “mammal” or “subject” or “patient” as used herein includes bothhumans and non-humans and include but is not limited to humans,non-human primates, canines, felines, murines, bovines, equines, andporcines.

The term percent “identity,” in the context of two or more nucleic acidor polypeptide sequences, refer to two or more sequences or subsequencesthat have a specified percentage of nucleotides or amino acid residuesthat are the same, when compared and aligned for maximum correspondence,as measured using one of the sequence comparison algorithms describedbelow (e.g., BLASTP and BLASTN or other algorithms available to personsof skill) or by visual inspection. Depending on the application, thepercent “identity” can exist over a region of the sequence beingcompared, e.g., over a functional domain, or, alternatively, exist overthe full length of the two sequences to be compared.

For sequence comparison, typically one sequence acts as a referencesequence to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv Appl Math 1981;2:482, by the homology alignment algorithm of Needleman & Wunsch, J MolBiol 1970; 48:443, by the search for similarity method of Pearson &Lipman, PNAS 1988; 85:2444, by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by visual inspection (see generally Ausubel et al, infra).

One example of an algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul et al., J Mol Biol 1990; 215:403-10. Softwarefor performing BLAST analyses is publicly available through the NationalCenter for Biotechnology Information website.

The term “sufficient amount” means an amount sufficient to produce adesired effect.

The term “therapeutically effective amount” is an amount that iseffective to ameliorate a symptom of a disease. A therapeuticallyeffective amount can be a “prophylactically effective amount” asprophylaxis can be considered therapy.

The term “about” will be understood by persons of ordinary skill andwill vary to some extent depending on the context in which it is used.If there are uses of the term which are not clear to persons of ordinaryskill given the context in which it is used, “about” will mean up toplus or minus 10% of the particular value.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an” and “the” include plural referentsunless the context clearly dictates otherwise.

Optimized Binuclease Fusion Proteins

The optimized binuclease fusion proteins of the disclosure include an Fcdomain, or a variant or fragment thereof, that alters the serumhalf-life of the nuclease molecules to which it is fused compared tonuclease molecules that are not fused to the Fc domain, or a variant orfragment thereof.

In some embodiments, a composition of the disclosure includes anoptimized binuclease fusion protein. In some embodiments, an optimizedbinuclease fusion protein includes a nuclease domain operably coupled toan Fc domain, or a variant or fragment thereof.

In some embodiments, the nuclease domain is operably coupled to the Fcdomain, or a variant or fragment thereof, via a linker domain. In someembodiments, the linker domain is a linker peptide. In some embodiments,the linker domain is a linker nucleotide.

In some embodiments, the optimized binuclease fusion protein includes aleader molecule, e.g., a leader peptide. In some embodiments, the leadermolecule is a leader peptide positioned at the N-terminus of thenuclease domain. In some embodiments, an optimized binuclease fusionprotein of the invention comprises a leader peptide at the N-terminus ofthe molecule, wherein the leader peptide is later cleaved from theoptimized binuclease fusion protein. Methods for generating nucleic acidsequences encoding a leader peptide fused to a recombinant protein arewell known in the art. In some embodiments, any of the optimizedbinuclease fusion proteins of the present invention can be expressedeither with or without a leader fused to their N-terminus. The proteinsequence of an optimized binuclease fusion protein of the presentdisclosure following cleavage of a fused leader peptide can be predictedand/or deduced by one of skill in the art.

In some embodiments the leader is a VK3 leader peptide (VK3LP), whereinthe leader peptide is fused to the N-terminus of the optimizedbinuclease fusion protein. Such leader sequences can improve the levelof synthesis and secretion of the optimized binuclease fusion protein inmammalian cells. In some embodiments, the leader is cleaved, yieldingoptimized binuclease fusion proteins. In some embodiments, an optimizedbinuclease fusion protein of the present invention is expressed withouta leader peptide fused to its N-terminus, and the resulting optimizedbinuclease fusion protein has an N-terminal methionine.

In some embodiments, the optimized binuclease fusion protein includestwo nuclease domains operably coupled to each other in tandem andfurther operably coupled to the N- or C-terminus of the same ordifferent Fc domains, or a variant or fragment thereof.

FIG. 1 displays exemplary configurations of the optimized binucleasefusion proteins, and the Sequence Table provides the sequences ofexemplary optimized binuclease fusion proteins of variousconfigurations.

In some embodiments, an optimized binuclease fusion protein is amulti-nuclease protein (e.g., both RNase and DNase or two RNA or DNAnucleases with different specificity for substrate) fused to the same ordifferent Fc domains, or a variant or fragment thereof, thatspecifically binds to extracellular immune complexes.

In one embodiment, the nuclease domain is operably coupled (e.g.,chemically conjugated or genetically fused (e.g., either directly or viaa polypeptide linker)) to the N-terminus of a Fc domain, or a variant orfragment thereof. In another embodiment, the nuclease domain is operablycoupled (e.g., chemically conjugated or genetically fused (e.g., eitherdirectly or via a polypeptide linker)) to the C-terminus of a Fc domain,or a variant or fragment thereof. In other embodiments, a nucleasedomain is operably coupled (e.g., chemically conjugated or geneticallyfused (e.g., either directly or via a polypeptide linker)) via an aminoacid side chain of a Fc domain, or a variant or fragment thereof.

In certain embodiments, the optimized binuclease fusion proteins of thedisclosure comprise two or more nuclease domains and at least one Fcdomain, or a variant or fragment thereof. For example, nuclease domainsmay be operably coupled to both the N-terminus and C-terminus of thesame or different Fc domains, or variants or fragments thereof, withoptional linkers between the nuclease domains and the Fc domain(s),variant(s) or fragment(s) thereof. In some embodiments, the nucleasedomains are identical, e.g., RNase and RNase, or DNase1 and DNase1. Inother embodiments, the nuclease domains are different, e.g., DNase andRNase.

In some embodiments, two or more nuclease domains are operably coupledto each other (e.g., via a polypeptide linker) in series, and the tandemarray of nuclease domains is operably coupled (e.g., chemicallyconjugated or genetically fused (e.g., either directly or via apolypeptide linker)) to either the C-terminus or the N-terminus of thesame or different Fc domains, or variants or fragments thereof. In otherembodiments, the tandem array of nuclease domains is operably coupled toboth the N-terminus and the C-terminus of the same Fc domain, or avariant or fragment thereof. In some embodiments, the nuclease domainsare operably linked in tandem (e.g., N-DNase-RNase-C or N-RNase-DNase-C)with or without a linker to the N- or C-terminus of the same ordifferent Fc domains. In some embodiments, the tandem binuclease fusionproteins form a homodimer or a heterodimer.

In other embodiments, one or more nuclease domains may be insertedbetween two Fc domains, or variants or fragments thereof. For example,one or more nuclease domains may form all or part of a polypeptidelinker of a optimized binuclease fusion protein of the disclosure.

In some embodiments, the optimized binuclease fusion proteins compriseat least two nuclease domains (e.g., RNase and DNase), at least onelinker domain, and at least one Fc domain, or a variant or fragmentthereof.

In some embodiments, the optimized binuclease fusion proteins of thedisclosure comprise a Fc domain, or a variant or fragment thereof, asdescribed supra, thereby increasing serum half-life and bioavailabilityof the optimized binuclease fusion proteins.

In some embodiments, an optimized binuclease fusion protein comprisesone or more polypeptides such as a polypeptide comprising an amino acidsequence as shown in any of SEQ ID NOs: 1-17.

It will be understood by the skilled artisan that other configurationsof the nuclease domains and Fc domains are possible, with the inclusionof optional linkers between the nuclease domains and/or between thenuclease domains and Fc domain. It will also be understood that domainorientation can be altered, so long as the nuclease domains are activein the particular configuration tested.

In certain embodiments, the optimized binuclease fusion proteins of thedisclosure have at least one nuclease domain specific for a targetmolecule which mediates a biological effect. In another embodiment,binding of the optimized binuclease fusion proteins of the disclosure toa target molecule (e.g. DNA or RNA) results in the reduction orelimination of the target molecule, e.g., from a cell, a tissue, or fromcirculation.

In other embodiments, the optimized binuclease fusion proteins of thedisclosure may be assembled together or with other polypeptides to formbinding proteins having two or more polypeptides (“multimers”), whereinat least one polypeptide of the multimer is an optimized binucleasefusion protein of the invention. Exemplary multimeric forms includedimeric, trimeric, tetrameric, and hexameric altered binding proteinsand the like. In one embodiment, the polypeptides of the multimer arethe same (i.e., homomeric altered binding proteins, e.g., homodimers,homotetramer). In another embodiment, the polypeptides of the multimerare different (e.g., heteromeric).

In some embodiments, an optimized binuclease fusion protein has a serumhalf-life that is increased at least about 1.5-fold, such as at least3-fold, at least 5-fold, at least 10-fold, at least about 20-fold, atleast about 50-fold, at least about 100-fold, at least about 200-fold,at least about 300-fold, at least about 400-fold, at least about500-fold, at least about 600-fold, at least about 700-fold, at leastabout 800-fold, at least about 900-fold, at least about 1000-fold, or1000-fold or greater relative to the corresponding nuclease moleculesnot fused to the Fc domain, or a variant or fragment thereof. In otherembodiments, an optimized binuclease fusion protein has a serumhalf-life that is decreased at least about 1.5-fold, such as at least3-fold, at least 5-fold, at least 10-fold, at least about 20-fold, atleast about 50-fold, at least about 100-fold, at least about 200-fold,at least about 300-fold, at least about 400-fold, at least about500-fold, or 500-fold or lower relative to the corresponding nucleasemolecules not fused to the Fc domain, or a variant or fragment thereof.Routine art-recognized methods can be used to determine the serumhalf-life of optimized binuclease fusion proteins of the disclosure.

In some embodiments, the activity of the RNase in the optimizedbinuclease fusion protein is not less than about 10-fold less, such as9-fold less, 8-fold less, 7-fold less, 6-fold less, 5-fold less, 4-foldless, 3-fold less, or 2-fold less than the activity of a control RNasemolecule. In some embodiments, the activity of the RNase in theoptimized binuclease fusion protein is about equal to the activity of acontrol RNase molecule.

In some embodiments, the activity of the DNase in the optimizedbinuclease fusion protein is not less than about 10-fold less, such as9-fold less, 8-fold less, 7-fold less, 6-fold less, 5-fold less, 4-foldless, 3-fold less, or 2-fold less than the activity of a control DNasemolecule. In some embodiments, the activity of the DNase in theoptimized binuclease fusion protein is about equal to the activity of acontrol DNase molecule.

In some embodiments, the optimized binuclease fusion proteins can beactive towards extracellular immune complexes containing DNA and/or RNA,e.g., either in soluble form or deposited as insoluble complexes.

In some embodiments, the activity of the optimized binuclease fusionprotein is detectable in vitro and/or in vivo. In some embodiments, theoptimized binuclease fusion protein binds to a cell, a malignant cell,or a cancer cell and interferes with its biologic activity.

In another aspect, a multifunctional RNase or DNase molecule is providedthat is attached to another enzyme or antibody having bindingspecificity, such as an scFv targeted to RNA or DNA or a second nucleasedomain with the same or different specificities as the first domain.

In some embodiments, linker domains include (gly4ser) 3, 4 or 5 variantsthat alter the length of the linker by 5 amino acid progressions. Inanother embodiment, a linker domain is approximately 18 amino acids inlength and includes an N-linked glycosylation site, which can besensitive to protease cleavage in vivo. In some embodiments, an N-linkedglycosylation site can protect the optimized binuclease fusion proteinsfrom cleavage in the linker domain. In some embodiments, an N-linkedglycosylation site can assist in separating the folding of independentfunctional domains separated by the linker domain.

In some embodiments, the linker domain is an NLG linker(VDGASSPVNVSSPSVQDI) (SEQ ID NO: 41).

In some embodiments, the optimized binuclease fusion protein includessubstantially all or at least an enzymatically active fragment of aDNase. In some embodiments, the DNase is a Type I secreted DNase,preferably a human DNase such as mature human pancreatic DNase 1(UniProtKB entry P24855, SEQ ID NO: 20). In some embodiments, anaturally occurring variant allele, A114F (SEQ ID NO: 21), which showsreduced sensitivity to actin is included in a DNase1 optimizedbinuclease fusion protein (see Pan et al., JBC 1998; 273:18374-81; Zhenet al., BBRC 1997; 231:499-504; Rodriguez et al., Genomics 1997;42:507-13). In other embodiments, a naturally occurring variant allele,G105R (SEQ ID NO: 22), which exhibits high DNase activity relative towild type DNase1, is included in a DNase1 optimized binuclease fusionprotein (see Yasuda et al., Int J Biochem Cell Biol 2010; 42:1216-25).In some embodiments, this mutation is introduced into an optimizedbinuclease fusion protein to generate a more stable derivative of humanDNase 1. In some embodiments, the DNase is human, wild type DNase1 orhuman, DNase1 A114F mutated to remove all potential N-linkedglycosylation sites, i.e., asparagine residues at positions 18 and 106of the DNase1 domain set forth in SEQ ID NO: 20 (i.e., human DNase1N18S/N106S/A114F, SEQ ID NO: 24), which correspond to asparagineresidues at positions 40 and 128, respectively, of full lengthpancreatic DNase1 with the native leader (SEQ ID NO: 23). In someembodiments, the DNase is a human DNase1 comprising one or more basic(i.e., positively charged) amino acid substitutions to increase DNasefunctionality and chromatin cleavage. In some embodiments, basic aminoacids are introduced into human DNase1 at the DNA binding interface toenhance binding with negatively charged phosphates on DNA substrates(see U.S. Pat. Nos. 7,407,785; 6,391,607). This hyperactive DNase1 maybe referred to as “chromatin cutter.”

In some embodiments, 1, 2, 3, 4, 5 or 6 basic amino acid substitutionsare introduced into DNase1. For example, one or more of the followingresidues is mutated to enhance DNA binding: Gln9, Glu13, Thrl4, His44,Asn74, Asn110, Thr205. In some embodiments one or more of the foregoingamino acids are substituted with basic amino acids such as, arginine,lysine and/or histidine. For example, a mutant human DNase can includeone or more of the following substitutions: Q9R, E13R, T14K, H44K, N74K,N110R, T205K. In some embodiments, the mutant human DNase1 also includesan A114F substitution, which reduces sensitivity to actin (see U.S. Pat.No. 6,348,343). In one embodiment, the mutant human DNase1 includes thefollowing substitutions: E13R, N74K, A114F and T205K.

In some embodiments, the mutant human DNase1 further includes mutationsto remove potential glycosylation sites, e.g., asparagine residues atpositions 18 and 106 of the DNase1 domain set forth in SEQ ID NO: 20,which correspond to asparagines residues at positions 40 and 128,respectively of full length pancreatic DNase1 with the native leader. Inone embodiment, the mutant human DNase1 includes the followingsubstitutions: E13R/N74K/A114F/T205K/N18S/N106S.

In some embodiments, the DNase is DNase 1-like (DNaseL) enzyme, 1-3(UniProtKB entry Q13609; SEQ ID NO: 46). In some embodiments, the DNaseis three prime repair exonuclease 1 (TREX1; UniProtKB entry Q9NSU2; SEQID NO: 47). In some embodiments, the DNase is DNase2. In someembodiments, the DNase2 is DNAse2 alpha (i.e., DNase2; UnitProtKB entryO00115SEQ ID NO: 48) or DNase2 beta (i.e., DNase2-like acid DNase;UnitProtKB entry Q8WZ79; SEQ ID NO: 49). In some embodiments, theN-linked glycosylation sites of DNase 1L3, TREX1, DNase2 alpha, orDNase2 beta are mutated such as to remove potential N-linkedglycosylation sites. In some embodiments, a DNase-linker-Fc domaincontaining a 20 or 25 aa linker domain is made.

In some embodiments, the optimized binuclease fusion protein includes aRNase1, preferably human pancreatic RNase1 (UniProtKB entry P07998; SEQID NO: 27) of the RNase A family. In some embodiments, the human RNase1is mutated to remove all potential N-linked glycosylation sites, i.e.,asparagine residues at positions 34, 76, and 88 of the RNase1 domain setforth in SEQ ID NO: 27 (human RNase1 N34S/N76S/N88S, SEQ ID NO: 28),which correspond to asparagine residues at positions 62, 104, and 116,respectively, of full length pancreatic RNase1 with the native leader(SEQ ID NO: 29). In some embodiments, a RNase1-linker-Fc containing a 20or 25 aa linker domain is made.

In some embodiments, optimized binuclease fusion proteins includeDNase-linker-RNase-Fc, wherein the RNase1 domain is located at the COOHside of the Fc. In other embodiments, optimized binuclease fusionproteins include DNase-linker-RNase-Fc, wherein the RNase1 domain islocated at the NH2 side of the Fc. In some embodiments, optimizedbinuclease fusion proteins include: DNase-Fc and RNase-Fc;DNase1-Fc-linker-RNase and Fc domain; DNase1-Fc and Fc-linker-RNase;Fc-linker-DNase1 and Fc-linker-RNase; RNase-Fc-linker-DNase and Fcdomain; Fc-linker-DNase and Rnase-Fc; and RNase-Fc-linker-DNase.

In some embodiments, fusion junctions between enzyme domains and theother domains of the optimized binuclease fusion protein is optimized.

In some embodiments, the targets of the RNase enzyme activity ofoptimized binuclease fusion proteins are primarily extracellular,consisting of, e.g., RNA contained in immune complexes with anti-RNPautoantibody and RNA expressed on the surface of cells undergoingapoptosis. In some embodiments, the optimized binuclease fusion proteinis active in the acidic environment of the endocytic vesicles. In someembodiments, an optimized binuclease fusion protein including a Fcdomain, or a variant or fragment thereof, is adapted to be active bothextracellularly and in the endocytic environment. In some aspects, thisallows an optimized binuclease fusion protein including a wild-type Fcdomain, or a variant or fragment thereof, to stop TLR7 signaling throughpreviously engulfed immune complexes or by RNAs that activate TLR7 afterviral infection. In some embodiments, the wild type RNase of anoptimized binuclease fusion protein is not resistant to inhibition by anRNase cytoplasmic inhibitor. In some embodiments, the wild type RNase ofan optimized binuclease fusion protein is not active in the cytoplasm ofa cell.

In some embodiments, optimized binuclease fusion proteins include bothDNase and RNase. In some embodiments, these optimized binuclease fusionproteins improve therapy of SLE because they digest or degrade immunecomplexes containing RNA, DNA, or a combination of both RNA and DNA, andare active extracellularly.

Fc Domain

In some embodiments, the polypeptide comprising one or more nucleasedomains is operably coupled to a Fc domain, which serves as a scaffoldas well as a means to increase the serum half-life of the polypeptide.In some embodiments, the one or more nuclease domains and/or the Fcdomain is aglycosylated, deglycosylated, or underglycosylated.

Suitable Fc domains are well-known in the art and include, but are notlimited to, Fc and Fc variants, such as those disclosed inWO2011/053982, WO 02/060955, WO 02/096948, WO05/047327, WO05/018572, andUS 2007/0111281 (the contents of the foregoing are incorporated hereinby reference). It is within the abilities of the skilled artisan to useroutine methods to introduce Fc domains (e.g., cloning, conjugation)into the optimized binuclease fusion proteins disclosed herein (with orwithout altered glycosylation).

In some embodiments, the Fc domain is a wild type human IgG1 Fc, such asis shown in SEQ ID NO: 45.

In some embodiments, an Fc domain is altered or modified, e.g., bymutation which results in an amino acid addition, deletion, orsubstitution. As used herein, the term “Fc domain variant” refers to anFc domain having at least one amino acid modification, such as an aminoacid substitution, as compared to the wild-type Fc from which the Fcdomain is derived. For example, wherein the Fc domain is derived from ahuman IgG1 antibody, a variant comprises at least one amino acidmutation (e.g., substitution) as compared to a wild type amino acid atthe corresponding position of the human IgG1 Fc region. The amino acidsubstitution(s) of an Fc variant may be located at a position within theFc domain referred to as corresponding to the position number that thatresidue would be given in an Fc region in an antibody (numberingaccording to EU index).

In one embodiment, the Fc variant comprises one or more amino acidsubstitutions at an amino acid position(s) located in a hinge region orportion thereof. In another embodiment, the Fc variant comprises one ormore amino acid substitutions at an amino acid position(s) located in aCH2 domain or portion thereof. In another embodiment, the Fc variantcomprises one or more amino acid substitutions at an amino acidposition(s) located in a CH3 domain or portion thereof. In anotherembodiment, the Fc variant comprises one or more amino acidsubstitutions at an amino acid position(s) located in a CH4 domain orportion thereof.

In some embodiments, the Fc region has a mutation at N83 (i.e., N297 byKabat numbering), yielding an aglycosylated Fc region (e.g., Fc N83S;SEQ ID NO: 50). In some embodiments, the Fc domain includes mutations inone or more of the three hinge region cysteines (residues 220, 226, and229, numbering according to the EU index). In some embodiments, one ormore of the three hinge cysteines in the Fc domain can be mutated to SCC(SEQ ID NO: 51) or SSS (SEQ ID NO: 52), where in “S” represents an aminoacid substitution of cysteine with serine. Accordingly “SCC” indicatesan amino acid substitution to serine of only the first cysteine of thethree hinge region cysteines (residues 220, 226, and 229, numberingaccording to the EU index), whereas “SSS” indicates that all threecysteines in the hinge region are substituted with serine (residues 220,226, and 229, numbering according to the EU index).

In some aspects, the Fc domain is a mutant human IgG1 Fc domain. In someaspects, a mutant Fc domain comprises one or more mutations in thehinge, CH2, and/or CH3 domains.

CH2 Substitutions

In some aspects, a mutant Fc domain includes a P238S mutation. In someaspects, a mutant Fc domain includes a P331S mutation. In some aspects,a mutant Fc domain includes a P238S mutation and a P331S mutation. Insome aspects, a mutant Fc domain comprises P238S and/or P331S, and mayinclude mutations in one or more of the three hinge cysteines (residues220, 226, and 229), numbering according to the EU index. In someaspects, a mutant Fc domain comprises P238S and/or P331S, and/or one ormore mutations in the three hinge cysteines (residues 220, 226, and229), numbering according to the EU index. In some aspects, a mutant Fcdomain comprises P238S and/or P331S, and/or mutations in a hingecysteine to SCC or in the three hinge cysteines to SSS. In some aspects,a mutant Fc domain comprises P238S and P331S and mutations in at leastone of the three hinge cysteines. In some aspects, a mutant Fc domaincomprises P238S and P331S and SCC. In some aspects, a mutant Fc domaincomprises P238S and P331S and SSS. In some aspects, a mutant Fc domainincludes P238S and SCC or SSS. In some aspects, a mutant Fc domainincludes P331S and SCC or SSS. (All numbering according to the EUindex).

In some aspects, a mutant Fc domain includes a mutation at a site ofN-linked glycosylation, such as N297, e.g., a substitution of asparaginefor another amino acid such as serine, e.g., N297S. In some aspects, amutant Fc domain includes a mutation at a site of N-linkedglycosylation, such as N297, e.g., a substitution of asparagine foranother amino acid such as serine, e.g., N297S and a mutation in one ormore of the three hinge cysteines. In some aspects, a mutant Fc domainincludes a mutation at a site of N-linked glycosylation, such as N297,e.g., a substitution of asparagine for another amino acid such asserine, e.g., N297S and mutations in one of the three hinge cysteines toSCC or all three cysteines to SSS. In some aspects, a mutant Fc domainincludes a mutation at a site of N-linked glycosylation, such as N297,e.g., a substitution of asparagine for another amino acid such asserine, e.g., N297 and one or more mutations in the CH2 domain whichdecrease FcγR binding and/or complement activation, such as mutations atP238 or P331 or both, e.g., P238S or P331S or both P238S and P331S. Insome aspects, such mutant Fc domains can further include a mutation inthe hinge region, e.g., SCC or SSS. (All numbering according to the EUindex.) In some aspects, the mutant Fc domain is as shown in theSequence Table or Sequence Listing herein.

CH3 Substitutions

Heterodimers can be preferentially formed by mutations in the CH3 domainof the Fc domain on the heterodimeric binuclease fusion proteinsdisclosed herein. Heavy chains were first engineered forheterodimerization using a “knobs-into-holes” strategy (Rigway B, etal., Protein Eng., 9 (1996) pp. 617-621). The term “knob-into-hole”refers to the technology directing the pairing of two polypeptidestogether in vitro or in vivo by introducing a pertuberance (knob) intoone polypeptide and a cavity (hole) into the other polypeptide at aninterface in which they interact. See e.g., WO 96/027011, WO 98/050431,U.S. Pat. No. 5,731,168, US2007/0178552, WO2009089004, US 20090182127.In particular, a combination of mutations in the CH3 domain can be usedto preferentially form heterodimers, for example, S354C, T366W in the“knob” heavy chain, and Y349C, T366S, L368A, Y407V in the “hole” heavychain. In some embodiments, the heterodimeric binuclease fusion proteindisclosed herein includes a first CH3 domain having the knob mutationT366W and a second CH3 domain having the hole mutations T366S, L368A,and Y407V. (Numbering according to the EU index.)

In some embodiments, the CH3 mutations are those described by Zymeworks(US 2012/0149876 A1, incorporated herein by reference; and VonKrcudenstein, T. S. et al. mAB3s, 5 (2013), pp. 646-654) and include thefollowing mutations: T350V, L351Y, F405A, and Y407V (first CH3 domain);and T350V, T366L, K392L, T394W (second CH3 domain). In some embodiments,the heterodimeric binuclease fusion protein disclosed herein includes afirst CH3 domain having T350V, L351Y, F405A, and Y407V mutations and asecond CH3 domain having T350V, T366L, K392L, T394W mutations.(Numbering according to the EU index.)

In some embodiments, the CH3 mutations are those described by Moore, G.L. et al. (mABs, 3 (2011), pp. 546-557) and include the followingmutations: S364H and F405A (first CH3 domain); and Y349T and T394F(second CH3 domain). In some embodiments, the heterodimeric binucleasefusion protein disclosed herein includes a first CH3 domain having S364Hand F405A mutations and a second CH3 domain having Y349T and T394Fmutations. (Numbering according to the EU index.)

In some embodiments, the CH3 mutations are those described byGunasekaran, K. et al. (J. Biol. Chem., 285 (2010), pp. 19637-19646) andinclude the following mutations: K409D and K392D (first CH3 domain); andD399K and E365K (second CH3 domain). In some embodiments, theheterodimeric binuclease fusion protein disclosed herein includes afirst CH3 domain having K409D and K392D mutations and a second CH3domain having D399K and E365K mutations. (Numbering according to the EUindex.)

The optimized binuclease fusion proteins of the disclosure may employart-recognized Fc variants which are known to impart an alteration ineffector function and/or FcR binding. For example, a change (e.g., asubstitution) at one or more of the amino acid positions disclosed inInternational PCT Publications WO88/07089A1, WO96/14339A1, WO98/05787A1,WO98/23289A1, WO99/51642A1, WO99/58572A1, WO00/09560A2, WO00/32767A1,WO00/42072A2, WO02/44215A2, WO02/060919A2, WO03/074569A2, WO04/016750A2,WO04/029207A2, WO04/035752A2, WO04/063351 A2, WO04/074455A2,WO04/099249A2, WO05/040217A2, WO04/044859, WO05/070963A1, WO05/077981A2,WO05/092925A2, WO05/123780A2, WO06/019447A1, WO06/047350A2, andWO06/085967A2; US Patent Publication Nos. US2007/0231329,US2007/0231329, US2007/0237765, US2007/0237766, US2007/0237767,US2007/0243188, US20070248603, US20070286859, US20080057056; or U.S.Pat. Nos. 5,648,260; 5,739,277; 5,834,250; 5,869,046; 6,096,871;6,121,022; 6,194,551; 6,242,195; 6,277,375; 6,528,624; 6,538,124;6,737,056; 6,821,505; 6,998,253; 7,083,784; and 7,317,091, each of whichis incorporated by reference herein. In one embodiment, the specificchange (e.g., the specific substitution of one or more amino acidsdisclosed in the art) may be made at one or more of the disclosed aminoacid positions. In another embodiment, a different change at one or moreof the disclosed amino acid positions (e.g., the different substitutionof one or more amino acid position disclosed in the art) may be made.

Other amino acid mutations in the Fc domain are contemplated to reducebinding to the Fc gamma receptor and Fc gamma receptor subtypes. Theassignment of amino acids residue numbers to an Fc domain is inaccordance with the definitions of Kabat. See, e.g., Sequences ofProteins of Immunological Interest (Table of Contents, Introduction andConstant Region Sequences sections), 5th edition, Bethesda, Md.: NIHvol. 1:647-723 (1991); Kabat et al., “Introduction” Sequences ofProteins of Immunological Interest, US Dept of Health and HumanServices, NIH, 5th edition, Bethesda, Md. vol. 1:xiii-xcvi (1991);Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987); Chothia et al., Nature342:878-883 (1989), each of which is herein incorporated by referencefor all purposes.”

For example, mutations at positions 238, 239, 248, 249, 252, 254, 255,256, 258, 265, 267, 268, 269, 270, 272, 279, 280, 283, 285, 298, 289,290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 312, 315, 322,324, 327, 329, 330, 331, 333, 334, 335, 337, 338, 340, 356, 360, 373,376, 378, 379, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437,438 or 439 of the Fc region can alter binding as described in U.S. Pat.No. 6,737,056, issued May 18, 2004, incorporated herein by reference inits entirety. This patent reported that changing Pro331 in IgG3 to Serresulted in six fold lower affinity as compared to unmutated IgG3,indicating the involvement of Pro331 in Fc gamma RI binding. Inaddition, amino acid modifications at positions 234, 235, 236, and 237,297, 318, 320 and 322 are disclosed as potentially altering receptorbinding affinity in U.S. Pat. No. 5,624,821, issued Apr. 29, 1997 andincorporated herein by reference in its entirety. (Numbering accordingto the EU index.)

Further mutations contemplated for use include, e.g., those described inU.S. Pat. App. Pub. No. 2006/0235208, published Oct. 19, 2006 andincorporated herein by reference in its entirety. This publicationsdescribe Fc variants that exhibit reduced binding to Fc gamma receptors,reduced antibody dependent cell-mediated cytotoxicity, or reducedcomplement dependent cytotoxicity, that comprise at least one amino acidmodification in the Fc region, including 232G, 234G, 234H, 235D, 235G,235H, 2361, 236N, 236P, 236R, 237K, 237L, 237N, 237P, 238K, 239R, 265G,267R, 269R, 270H, 297S, 299A, 299I, 299V, 325A, 325L, 327R, 328R, 329K,330I, 330L, 330N, 330P, 330R, and 331L (numbering is according to the EUindex), as well as double mutants 236R/237K, 236R/325L, 236R/328R,237K/325L, 237K/328R, 325L/328R, 235G/236R, 267R/269R, 234G/235G,236R/237K/325L, 236R/325L/328R, 235G/236R/237K, and 237K/325L/328R.Other mutations contemplated for use as described in this publicationinclude 227G, 234D, 234E, 234G, 234I, 234Y, 235D, 235I, 235S, 236S,239D, 246H, 255Y, 258H, 260H, 2641, 267D, 267E, 268D, 268E, 272H, 272I,272R, 281D, 282G, 283H, 284E, 293R, 295E, 304T, 324G, 324I, 327D, 327A,328A, 328D, 328E, 328F, 328I, 328M, 328N, 328Q, 328T, 328V, 328Y, 330I,330L, 330Y, 332D, 332E, 335D, an insertion of G between positions 235and 236, an insertion of A between positions 235 and 236, an insertionof S between positions 235 and 236, an insertion of T between positions235 and 236, an insertion of N between positions 235 and 236, aninsertion of D between positions 235 and 236, an insertion of V betweenpositions 235 and 236, an insertion of L between positions 235 and 236,an insertion of G between positions 235 and 236, an insertion of Abetween positions 235 and 236, an insertion of S between positions 235and 236, an insertion of T between positions 235 and 236, an insertionof N between positions 235 and 236, an insertion of D between positions235 and 236, an insertion of V between positions 235 and 236, aninsertion of L between positions 235 and 236, an insertion of G betweenpositions 297 and 298, an insertion of A between positions 297 and 298,an insertion of S between positions 297 and 298, an insertion of Dbetween positions 297 and 298, an insertion of G between positions 326and 327, an insertion of A between positions 326 and 327, an insertionof T between positions 326 and 327, an insertion of D between positions326 and 327, and an insertion of E between positions 326 and 327(numbering is according to the EU index). Additionally, mutationsdescribed in U.S. Pat. App. Pub. No. 2006/0235208 include 227G/332E,234D/332E, 234E/332E, 234Y/332E, 234I/332E, 234G/332E, 235I/332E,235S/332E, 235D/332E, 235E/332E, 236S/332E, 236A/332E, 236S/332D,236A/332D, 239D/268E, 246H/332E, 255Y/332E, 258H/332E, 260H/332E,2641/332E, 267E/332E, 267D/332E, 268D/332D, 268E/332D, 268E/332E,268D/332E, 268E/330Y, 268D/330Y, 272R/332E, 272H/332E, 283H/332E,284E/332E, 293R/332E, 295E/332E, 304T/332E, 324I/332E, 324G/332E,324I/332D, 324G/332D, 327D/332E, 328A/332E, 328T/332E, 328V/332E,328I/332E, 328F/332E, 328Y/332E, 328M/332E, 328D/332E, 328E/332E,328N/332E, 328Q/332E, 328A/332D, 328T/332D, 328V/332D, 328I/332D,328F/332D, 328Y/332D, 328M/332D, 328D/332D, 328E/332D, 328N/332D,328Q/332D, 330L/332E, 330Y/332E, 330I/332E, 332D/330Y, 335D/332E,239D/332E, 239D/332E/330Y, 239D/332E/330L, 239D/332E/330I,239D/332E/268E, 239D/332E/268D, 239D/332E/327D, 239D/332E/284E,239D/268E/330Y, 239D/332E/268E/330Y, 239D/332E/327A,239D/332E/268E/327A, 239D/332E/330Y/327A, 332E/330Y/268E/327A,239D/332E/268E/330Y/327A, Insert G>297-298/332E, Insert A>297-298/332E,Insert S>297-298/332E, Insert D>297-298/332E, Insert G>326-327/332E,Insert A>326-327/332E, Insert T>326-327/332E, Insert D>326-327/332E,Insert E>326-327/332E, Insert G>235-236/332E, Insert A>235-236/332E,Insert S>235-236/332E, Insert T>235-236/332E, Insert N>235-236/332E,Insert D>235-236/332E, Insert V>235-236/332E, Insert L>235-236/332E,Insert G>235-236/332D, Insert A>235-236/332D, Insert S>235-236/332D,Insert T>235-236/332D, Insert N>235-236/332D, Insert D>235-236/332D,Insert V>235-236/332D, and Insert L>235-236/332D (numbering according tothe EU index) are contemplated for use. The mutant L234A/L235A isdescribed, e.g., in U.S. Pat. App. Pub. No. 2003/0108548, published Jun.12, 2003 and incorporated herein by reference in its entirety. Inembodiments, the described modifications are included eitherindividually or in combination. (Numbering according to the EU index.)

Linker Domains

In some embodiments, an optimized binuclease fusion protein includes alinker domain. In some embodiments, an optimized binuclease fusionprotein includes a plurality of linker domains. In some embodiments, thelinker domain is a polypeptide linker. In certain aspects, it isdesirable to employ a polypeptide linker to fuse Fc, or a variant orfragment thereof, with one or more nuclease domains to form an optimizedbinuclease fusion protein.

In one embodiment, the polypeptide linker is synthetic. As used herein,the term “synthetic” with respect to a polypeptide linker includespeptides (or polypeptides) which comprise an amino acid sequence (whichmay or may not be naturally occurring) that is linked in a linearsequence of amino acids to a sequence (which may or may not be naturallyoccurring) (e.g., a Fc sequence) to which it is not naturally linked innature. For example, the polypeptide linker may comprise non-naturallyoccurring polypeptides which are modified forms of naturally occurringpolypeptides (e.g., comprising a mutation such as an addition,substitution or deletion) or which comprise a first amino acid sequence(which may or may not be naturally occurring). The polypeptide linkersof the invention may be employed, for instance, to ensure that Fc, or avariant or fragment thereof, is juxtaposed to ensure proper folding andformation of a functional Fc, or a variant or fragment thereof.Preferably, a polypeptide linker compatible with the instant inventionwill be relatively non-immunogenic and not inhibit any non-covalentassociation among monomer subunits of a binding protein.

In certain embodiments, the optimized binuclease fusion protein employsan NLG linker as set forth in SEQ ID NO: 41.

In certain embodiments, the optimized binuclease fusion proteins of thedisclosure employ a polypeptide linker to join any two or more domainsin frame in a single polypeptide chain. In one embodiment, the two ormore domains may be independently selected from any of the Fc domains,or variants or fragments thereof, or nuclease domains discussed herein.For example, in certain embodiments, a polypeptide linker can be used tofuse identical Fc fragments, thereby forming a homodimeric Fc region. Inother embodiments, a polypeptide linker can be used to fuse different Fcfragments, thereby forming a heterodimeric Fc region. In otherembodiments, a polypeptide linker of the invention can be used togenetically fuse the C-terminus of a first Fc fragment to the N-terminusof a second Fc fragment to form a complete Fc domain.

In one embodiment, a polypeptide linker comprises a portion of a Fcdomain, or a variant or fragment thereof. For example, in oneembodiment, a polypeptide linker can comprise a Fc fragment (e.g., C orN domain), or a different portion of a Fc domain or variant thereof.

In another embodiment, a polypeptide linker comprises or consists of agly-ser linker. As used herein, the term “gly-ser linker” refers to apeptide that consists of glycine and serine residues. An exemplarygly/ser linker comprises an amino acid sequence of the formula(Gly₄Ser)n, wherein n is a positive integer (e.g., 1, 2, 3, 4, or 5). Apreferred gly/ser linker is (Gly₄Ser)4. Another preferred gly/ser linkeris (Gly₄Ser)3. Another preferred gly/ser linker is (Gly₄Ser)5. Incertain embodiments, the gly-ser linker may be inserted between twoother sequences of the polypeptide linker (e.g., any of the polypeptidelinker sequences described herein). In other embodiments, a gly-serlinker is attached at one or both ends of another sequence of thepolypeptide linker (e.g., any of the polypeptide linker sequencesdescribed herein). In yet other embodiments, two or more gly-ser linkerare incorporated in series in a polypeptide linker.

In other embodiments, a polypeptide linker of the invention comprises abiologically relevant peptide sequence or a sequence portion thereof.For example, a biologically relevant peptide sequence may include, butis not limited to, sequences derived from an anti-rejection oranti-inflammatory peptide. Said anti-rejection or anti-inflammatorypeptides may be selected from the group consisting of a cytokineinhibitory peptide, a cell adhesion inhibitory peptide, a thrombininhibitory peptide, and a platelet inhibitory peptide. In a preferredembodiment, a polypeptide linker comprises a peptide sequence selectedfrom the group consisting of an IL-1 inhibitory or antagonist peptidesequence, an erythropoietin (EPO)-mimetic peptide sequence, athrombopoietin (TPO)-mimetic peptide sequence, G-CSF mimetic peptidesequence, a TNF-antagonist peptide sequence, an integrin-binding peptidesequence, a selectin antagonist peptide sequence, an anti-pathogenicpeptide sequence, a vasoactive intestinal peptide (VIP) mimetic peptidesequence, a calmodulin antagonist peptide sequence, a mast cellantagonist, a SH3 antagonist peptide sequence, an urokinase receptor(UKR) antagonist peptide sequence, a somatostatin or cortistatin mimeticpeptide sequence, and a macrophage and/or T-cell inhibiting peptidesequence. Exemplary peptide sequences, any one of which may be employedas a polypeptide linker, are disclosed in U.S. Pat. No. 6,660,843, whichis incorporated by reference herein.

Other linkers that are suitable for use in optimized binuclease fusionproteins are known in the art, for example, the serine-rich linkersdisclosed in U.S. Pat. No. 5,525,491, the helix forming peptide linkers(e.g., A(EAAAK)nA (n=2-5)) disclosed in Arai et al., Protein Eng 2001;14:529-32, and the stable linkers disclosed in Chen et al., Mol Pharm2011; 8:457-65, i.e., the dipeptide linker LE, a thrombin-sensitivedisulfide cyclopeptide linker, and the alpha-helix forming linkerLEA(EAAAK)₄ALEA(EAAAK)₄ALE (SEQ ID NO: 53).

Other exemplary linkers include GS linkers (i.e., (GS)n), GGSG (SEQ IDNO: 70) linkers (i.e., (GGSG)n), GSAT linkers (SEQ ID NO: 44), SEGlinkers, and GGS linkers (i.e., (GGSGGS)n), wherein n is a positiveinteger (e.g., 1, 2, 3, 4, or 5). Other suitable linkers for use in theoptimized binuclease fusion proteins can be found using publiclyavailable databases, such as the Linker Database(ibi.vu.nl/programs/linkerdbwww). The Linker Database is a database ofinter-domain linkers in multi-functional enzymes which serve aspotential linkers in novel fusion proteins (see, e.g., George et al.,Protein Engineering 2002; 15:871-9).

It will be understood that variant forms of these exemplary polypeptidelinkers can be created by introducing one or more nucleotidesubstitutions, additions or deletions into the nucleotide sequenceencoding a polypeptide linker such that one or more amino acidsubstitutions, additions or deletions are introduced into thepolypeptide linker. Mutations may be introduced by standard techniques,such as site-directed mutagenesis and PCR-mediated mutagenesis.

Polypeptide linkers of the disclosure are at least one amino acid inlength and can be of varying lengths. In one embodiment, a polypeptidelinker of the invention is from about 1 to about 50 amino acids inlength. As used in this context, the term “about” indicates+/− two aminoacid residues. Since linker length must be a positive integer, thelength of from about 1 to about 50 amino acids in length, means a lengthof from 1 to 48-52 amino acids in length. In another embodiment, apolypeptide linker of the disclosure is from about 10-20 amino acids inlength. In another embodiment, a polypeptide linker of the disclosure isfrom about 15 to about 50 amino acids in length.

In another embodiment, a polypeptide linker of the disclosure is fromabout 20 to about 45 amino acids in length. In another embodiment, apolypeptide linker of the disclosure is from about 15 to about 25 aminoacids in length. In another embodiment, a polypeptide linker of thedisclosure is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, or 61 or more amino acids in length.

Polypeptide linkers can be introduced into polypeptide sequences usingtechniques known in the art. Modifications can be confirmed by DNAsequence analysis. Plasmid DNA can be used to transform host cells forstable production of the polypeptides produced.

Exemplary Optimized Binuclease Fusion Proteins

The optimized binuclease fusion proteins of the invention are modular,and can be configured to incorporate various individual domains. Forexample, in one embodiment, the optimized binuclease fusion protein mayinclude the mutant, human DNase1 A114F domain set forth in (SEQ ID NO:21). In another embodiment, the optimized binuclease fusion protein mayinclude the mutant, human DNase1 N18S/N106S/A114F domain set forth inSEQ ID NO: 24. In another embodiment, the optimized binuclease fusionprotein may include the human, wild-type RNase1 domain set forth in SEQID NO: 27. In another embodiment, the optimized binuclease fusionprotein may include the human, mutant RNase1 N34S/N76S/N88S domain setforth in SEQ ID NO: 28. In another embodiment, the optimized binucleasefusion protein may include the (Gly₄Ser)3 linker domain set forth in SEQID NO: 30. In another embodiment, the optimized binuclease fusionprotein may include the NLG linker set forth in SEQ ID NO: 41. Inanother embodiment, the optimized binuclease fusion protein may includea VK3LP leader (SEQ ID NO: 54). It will be understood to the skilledartisan that these individual domains can be operably coupled to eachother in any order to form an optimized binuclease fusion protein thatis enzymatically active. For example, as detailed in the specificexamples below, RNase1 can be operably coupled to an Fc domain. Inanother example, RNase1 can be operatively coupled to Fc domain via a(Gly₄Ser)3 linker domain. In yet another example, DNase1 A114F can beoperatively coupled to Fc domain. In yet another example, DNase1 A114Fcan be operatively coupled to Fc domain via a (Gly₄Ser)3 linker domain.Various other configurations are possible, with non-limiting exemplaryconfigurations disclosed herein, in FIG. 1 and in the Sequence Table.

In some embodiments, an optimized binuclease fusion protein comprises awild-type, human RNase1 domain operably coupled to a mutant Fc domaincomprising SCC hinge and CH2 mutations P238S and P331S, or fragmentthereof, and a mutated human DNase1 domain operably coupled to the humanRNase1, thereby forming a tandem homodimer. In some embodiments, theDNase1 is linked to the RNase1 via a peptide linker, such as an NLGlinker disclosed herein. In some embodiments, the RNase1 is operablylinked with or without a linker to the N-terminus of the Fc domain. Insome embodiments, an optimized binuclease fusion protein comprises apolypeptide having the amino acid sequence set forth in SEQ ID NO: 1. Insome embodiments, the RNase1 is operably linked with or without a linkerto the C-terminus of the Fc domain. In some embodiments, an optimizedbinuclease fusion protein comprises a polypeptide having the amino acidsequence set forth in SEQ ID NO: 2. In some embodiments, the optimizedbinuclease fusion protein is homodimeric or heterodimeric.

In some embodiments, an optimized binuclease fusion protein is aheterodimer comprising mutant human DNase 1 domain operably coupled withor without a linker to a first mutant Fc domain, having SCC hinge, CH2mutations P238S, P331S, and CH3 mutations T350V, L351Y, F405A and Y407V,or a variant or fragment thereof, and a wild-type human RNase 1 domainoperably coupled with or without a linker to a second mutant Fc domaincomprising SCC hinge, CH2 mutations P238S and P331S and CH3 mutationsT350V, T366L, K392L and T394W, or a fragment thereof. In someembodiments, the DNase1 and RNase 1 are both linked to the N-terminus oftheir respective Fc domains. In some embodiments, an optimizedbinuclease fusion protein is a heterodimer comprising a polypeptidecomprising the amino acid sequence set forth in SEQ ID NOs: 3 and apolypeptide comprising the amino acid sequence set for in SEQ ID NO: 4.

In some embodiments, an optimized binuclease fusion protein is aheterodimer comprising a mutant human DNase 1 domain and wild-type humanRNase 1 domain, both operably coupled with or without a linker to afirst mutant Fc domain, comprising SCC hinge, CH2 mutations P238S andP331S and CH3 mutations T350V, T366L, K392L and T394W, or a fragmentthereof, and a second mutant Fc domain, having mutations T350V, T366L,K392L and T394W, or fragment thereof. In some embodiments, the DNase1and RNase 1 are linked to the N-terminus and C-terminus, respectively,of the first and second Fc domains. In some embodiments, an optimizedbinuclease fusion protein is a heterodimer comprising a polypeptidecomprising the sequence set forth in SEQ ID NO: 5 and a polypeptidecomprising the amino acid sequence set forth in SEQ ID NO: 6.

In some embodiments, an optimized binuclease fusion protein is aheterodimer comprising a mutant human DNase 1 domain, operably coupledwith or without a linker to a mutant Fc domain comprising SCC hinge, CH2mutations P238S, P331S and CH3 mutations T350V, L351Y, F405A and Y407V,or fragment thereof, and wild-type human RNase 1 domain, operablycoupled with or without a linker to a mutant Fc domain comprising SCChinge, CH2 mutations P238S, P331S and CH3 mutations T350V, T366L, K392Land T394W, or fragment thereof. In some embodiments, the DNase1 islinked to the N-terminus of the Fc domain and the RNase1 is linked tothe C-terminus of the Fc domain. In some embodiments, an optimizedbinuclease fusion protein is a heterodimer comprising a polypeptidecomprising the sequence set forth in SEQ ID NO: 7 and a polypeptidecomprising the amino acid sequence set forth in SEQ ID NO: 8.

In some embodiments, an optimized binuclease fusion protein is aheterodimer comprising a mutant human DNase 1 domain operably coupledwith or without a linker to a mutant Fc domain comprising SCC hinge, CH2mutations P238S, P331S and CH3 mutations T350V, L351Y, F405A and Y407V,or fragment thereof, and a wild-type human RNase 1 domain operablycoupled with or without a linker to a mutant Fc domain comprising SCChinge, CH2 mutations P238S, P331S and CH3 mutations T350V, T366L, K392Land T394W, or fragment thereof. In some embodiments, the DNase1 andRNase 1 are both linked to the C-terminus of their respective Fcdomains. In some embodiments, an optimized binuclease fusion protein isa heterodimer comprising a polypeptide comprising the sequence set forthin SEQ ID NO: 9 and a polypeptide comprising the amino acid sequence setforth in SEQ ID NO: 10. In some embodiments, an optimized binucleasefusion protein is a heterodimer comprising a polypeptide comprising thesequence set forth in SEQ ID NO: 11 and a polypeptide comprising theamino acid sequence set forth in SEQ ID NO: 12.

In some embodiments, an optimized binuclease fusion protein is aheterodimer comprising a mutant human DNase 1 domain and wild-type humanRNase 1 domain, both operably coupled with or without a linker to amutant Fc domain comprising SCC hinge, CH2 mutations P238S, P331S andCH3 mutations T350V, L351Y, F405A and Y407V, or fragment thereof, and amutant Fc domain comprising SCC hinge, CH2 mutations P238S, P331S andCH3 mutations T350V, T366L, K392L and T394W, or fragment thereof. Insome embodiments, the DNase1 and RNase 1 are linked to the C-terminusand N-terminus, respectively, of the Fc domain. In some embodiments, anoptimized binuclease fusion protein is a heterodimer comprising apolypeptide comprising the sequence set forth in SEQ ID NO: 13 and apolypeptide comprising the amino acid sequence set forth in SEQ ID NO:14.

In some embodiments, an optimized binuclease fusion protein is aheterodimer comprising a mutant human DNase 1 domain, operably coupledwith or without a linker to a mutant Fc domain comprising SCC hinge, CH2mutations P238S, P331S and CH3 mutations T350V, L351Y, F405A and Y407V,or fragment thereof, and wild-type human RNase 1 domain, operablycoupled with or without a linker to a mutant Fc domain comprising SCChinge, CH2 mutations P238S, P331S and CH3 mutations T350V, T366L, K392Land T394W, or fragment thereof. In some embodiments, the DNase1 islinked to the C-terminus of the Fc domain and the RNase1 is linked tothe N-terminus of the Fc domain. In some embodiments, an optimizedbinuclease fusion protein is a heterodimer comprising a polypeptidecomprising the sequence set forth in SEQ ID NO: 15 and a polypeptidecomprising the amino acid sequence set forth in SEQ ID NO: 16.

In some embodiments, an optimized binuclease fusion protein comprising apolypeptide having an amino acid sequence at least 80% identical, suchas 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or at least99.5% identical to an amino acid sequence of any one of SEQ ID NOs:1-17. In some embodiments, the polypeptide comprises an amino acidsequence set for in any one of SEQ ID NOs: 1-17.

In some embodiments, the foregoing optimized binuclease fusion proteinshave a leader sequence.

It will be understood by one of ordinary skill that the leader andlinker sequences are optional and are not limited to those described inthe embodiments above. For example, the RNase and/or DNase domains canbe directly fused to the N- and/or C-terminus of Fc, or variant orfragment thereof; the leader domain can be any of those known in the artto be useful for its intended purpose, e.g., to increase proteinexpression and/or secretion (e.g., a Gaussia luciferase signal peptide(MGVKVLFALICIAVAEA; SEQ ID NO: 31)); the linker can be any linker knownin the art, e.g., (Gly₄Ser)n, NLG (VDGASSPVNVSSPSVQDI; SEQ ID NO: 41),LE, thrombin-sensitive disulphide cyclopeptide linker,LEA(EAAAK)₄ALEA(EAAAK)₄ (SEQ ID NO: 32), or an in vivo cleavabledisulphide linker, as described herein. It will also be understood thatit is within the abilities of a skilled artisan to make thecorresponding changes to the amino acid sequences of the optimizedbinuclease fusion protein using routine cloning and recombinationmethods. It will also be understood that the asparagine residues in thenuclease domains (i.e., N34, N76, and N88 in RNase1, and N18 and N106 inDNase1) can be substituted with an amino acid other than serine (e.g.,glutamine), as long as the amino acid does not serve as an acceptor forN-linked glycosylation.

Methods of Making Optimized Binuclease Fusion Proteins

The optimized binuclease fusion proteins of this disclosure largely maybe made in transformed or transfected host cells using recombinant DNAtechniques. To do so, a recombinant DNA molecule coding for the peptideis prepared. Methods of preparing such DNA molecules are well known inthe art. For instance, sequences coding for the peptides could beexcised from DNA using suitable restriction enzymes. Alternatively, theDNA molecule could be synthesized using chemical synthesis techniques,such as the phosphoramidate method. Also, a combination of thesetechniques could be used. The invention also includes a vector capableof expressing the peptides in an appropriate host. The vector comprisesthe DNA molecule that codes for the peptides operably coupled toappropriate expression control sequences. Methods of affecting thisoperative linking, either before or after the DNA molecule is insertedinto the vector, are well known. Expression control sequences includepromoters, activators, enhancers, operators, ribosomal nuclease domains,start signals, stop signals, cap signals, polyadenylation signals, andother signals involved with the control of transcription or translation.

The resulting vector having the DNA molecule thereon is used totransform or transfect an appropriate host. This transformation ortransfection may be performed using methods well known in the art.

Any of a large number of available and well-known host cells may be usedin the practice of this invention. The selection of a particular host isdependent upon a number of factors recognized by the art. These include,for example, compatibility with the chosen expression vector, toxicityof the peptides encoded by the DNA molecule, rate of transformation ortransfection, ease of recovery of the peptides, expressioncharacteristics, bio-safety and costs. A balance of these factors mustbe struck with the understanding that not all hosts may be equallyeffective for the expression of a particular DNA sequence. Within thesegeneral guidelines, useful microbial hosts include bacteria (such as E.coli), yeast (such as Saccharomyces) and other fungi, insects, plants,mammalian (including human) cells in culture, or other hosts known inthe art. In a preferred embodiment, the optimized binuclease fusionproteins are produced in CHO cells.

Next, the transformed or transfected host is cultured and purified. Hostcells may be cultured under conventional fermentation or cultureconditions so that the desired compounds are expressed. Suchfermentation and culture conditions are well known in the art. Finally,the peptides are purified from culture by methods well known in the art.

The compounds may also be made by synthetic methods. For example, solidphase synthesis techniques may be used. Suitable techniques are wellknown in the art, and include those described in Merrifield (1973),Chem. Polypeptides, pp. 335-61 (Katsoyannis and Panayotis eds.);Merrifield (1963), J. Am. Chem. Soc. 85: 2149; Davis et al., BiochemIntl 1985; 10: 394-414; Stewart and Young (1969), Solid Phase PeptideSynthesis; U.S. Pat. No. 3,941,763; Finn et al. (1976), The Proteins(3rd ed.) 2: 105-253; and Erickson et al. (1976), The Proteins (3rd ed.)2: 257-527. Solid phase synthesis is the preferred technique of makingindividual peptides since it is the most cost-effective method of makingsmall peptides. Compounds that contain derivatized peptides or whichcontain non-peptide groups may be synthesized by well-known organicchemistry techniques.

Other methods are of molecule expression/synthesis are generally knownin the art to one of ordinary skill.

Optimized Binuclease Fusion Proteins with Altered Glycosylation

Glycosylation (e.g., O-lined or N-linked glycosylation) can impact theserum half-life of the optimized binuclease fusion proteins of thedisclosure by, e.g., minimizing their removal from circulation bymannose and asialoglycoprotein receptors and other lectin-likereceptors. Accordingly, in some embodiments, the optimized binucleasefusion proteins of the disclosure are prepared in aglycosylated,deglycosylated, or underglycosylated form. Preferably, N-linkedglycosylation is altered and the optimized binuclease fusion protein isaglycosyated.

In some embodiments, all asparagine residues in an optimized binucleasefusion protein that conform to the Asn-X-Ser/Thr (X can be any othernaturally occurring amino acid except Pro) consensus are mutated toresidues that do not serve as acceptors of N-linked glycosylation (e.g.,serine, glutamine), thereby eliminating glycosylation of the optimizedbinuclease fusion protein when synthesized in a cell that glycosylatesproteins.

In some embodiments, optimized binuclease fusion proteins lackingN-linked glycosylation sites are produced in mammalian cells. In oneembodiment, the mammalian cell is a CHO cell. Accordingly, in a specificembodiment, an aglycosylated optimized binuclease fusion protein isproduced in a CHO cell.

In other embodiments, a reduction or lack of N-glycosylation is achievedby, e.g., producing optimized binuclease fusion proteins in a host(e.g., bacteria such as E. coli), mammalian cells engineered to lack oneor more enzymes important for glycosylation, or mammalian cells treatedwith agents that prevent glycosylation, such as tunicamycin (aninhibitor of Dol-PP-GlcNAc formation).

In some embodiments, the optimized binuclease fusion proteins areproduced in lower eukaryotes engineered to produce glycoproteins withcomplex N-glycans, rather than high mannose type sugars (see, e.g.,US2007/0105127).

In some embodiments, glycosylated optimized binuclease fusion proteins(e.g., those produced in mammalian cells such as CHO cells) are treatedchemically or enzymatically to remove one or more carbohydrate residues(e.g., one or more mannose, fucose, and/or N-acetylglucosamine residues)or to modify or mask one or more carbohydrate residues. Suchmodifications or masking may reduce binding of the optimized binucleasefusion proteins to mannose receptors, and/or asialoglycoproteinreceptors, and/or other lectin-like receptors. Chemical deglycosylationcan be achieved by treating an optimized binuclease fusion protein withtrifluoromethane sulfonic acid (TFMS), as disclosed in, e.g., Sojar etal., JBC 1989; 264:2552-9 and Sojar et al., Methods Enzymol 1987;138:341-50, or by treating with hydrogen fluoride, as disclosed in Sojaret al. (1987, supra). Enzymatic removal of N-linked carbohydrates fromoptimized binuclease fusion proteins can be achieved by treating anoptimized binuclease fusion protein with protein N-glycosidase (PNGase)A or F, as disclosed in Thotakura et al. (Methods Enzymol 1987;138:350-9). Other art-recognized commercially available deglycosylatingenzymes that are suitable for use includeendo-alpha-N-acetyl-galactosaminidase, endoglycosidase F1,endoglycosidase F2, endoglycosidase F3, and endoglycosidase H. In someembodiments, one or more of these enzymes can be used to deglycosylatethe optimized binuclease fusion proteins of the disclosure. Alternativemethods for deglycosylation are disclosed in, e.g., U.S. Pat. No.8,198,063.

In some embodiments, the optimized binuclease fusion proteins arepartially deglycosylated. Partial deglycosylation can be achieved bytreating the optimized binuclease fusion proteins with anendoglycosidase (e.g., endoglycosidase H), which cleaves N-linked highmannose carbohydrate but not complex type carbohydrates, leaving asingle GlcNAc residue linked to the asparagine. Optimized binucleasefusion proteins treated with endoglycosidase H will lack high mannosecarbohydrates, resulting in a reduced interaction with the hepaticmannose receptor. Although this receptor recognizes terminal GlcNAc, theprobability of a productive interaction with the single GlcNAc on theprotein surface is not as great as with an intact high mannosestructure.

In other embodiments, glycosylation of an optimized binuclease fusionprotein is modified, e.g., by oxidation, reduction, dehydration,substitution, esterification, alkylation, sialylation, carbon-carbonbond cleavage, or the like, to reduce clearance of the optimizedbinuclease fusion proteins from blood. In some embodiments, theoptimized binuclease fusion proteins are treated with periodate andsodium borohydride to modify the carbohydrate structure. Periodatetreatment oxidizes vicinal diols, cleaving the carbon-carbon bond andreplacing the hydroxyl groups with aldehyde groups; borohydride reducesthe aldehydes to hydroxyls. Many sugar residues include vicinal diolsand, therefore, are cleaved by this treatment. Prolonged serum half-lifewith periodate and sodium borohydride is exemplified by the sequentialtreatment of the lysosomal enzyme β-glucuronidase with these agents(see, e.g., Houba et al. (1996) Bioconjug Chem 1996:7:606-11; Stahl etal. PNAS 1976; 73:4045-9; Achord et al. Pediat. Res 1977; 11:816-22;Achord et al. Cell 1978; 15:269-78). A method for treatment withperiodate and sodium borohydride is disclosed in Hickman et al., BBRC1974; 57:55-61. A method for treatment with periodate andcyanoborohydride, which increases the serum half-life and tissuedistribution of ricin, is disclosed in Thorpe et al. Eur J Biochem 1985;147:197-206.

In one embodiment, the carbohydrate structures of an optimizedbinuclease fusion protein can be masked by addition of one or moreadditional moieties (e.g., carbohydrate groups, phosphate groups, alkylgroups, etc.) that interfere with recognition of the structure by amannose or asialoglycoprotein receptor or other lectin-like receptors.

In some embodiments, one or more potential glycosylation sites areremoved by mutation of the nucleic acid encoding the optimizedbinuclease fusion protein, thereby reducing glycosylation(underglycosylation) of the optimized binuclease fusion protein whensynthesized in a cell that glycosylates proteins, e.g., a mammalian cellsuch as a CHO cell. In some embodiments, it may be desirable toselectively underglycosylate the nuclease domain of the optimizedbinuclease fusion proteins by mutating the potential N-linkedglycosylation sites therein if, e.g., the underglycosylated optimizedbinuclease fusion protein exhibits increased activity or contributes toincreased serum half-life. In other embodiments, it may be desirable tounderglycosylate portions of theoptimized binuclease fusion protein suchthat regions other than the nuclease domain lack N-glycosylation if, forexample, such a modification improves the serum half-life of theoptimized binuclease fusion protein. Alternatively, other amino acids inthe vicinity of glycosylation acceptors can be modified, disrupting arecognition motif for glycosylation enzymes without necessarily changingthe amino acid that would normally be glycosylated.

In some embodiments, glycosylation of an optimized binuclease fusionprotein can be altered by introducing glycosylation sites. For example,the amino acid sequence of the optimized binuclease fusion protein canbe modified to introduce the consensus sequence for N-linkedglycosylation of Asp-X-Ser/Thr (X is any amino acid other than proline).Additional N-linked glycosylation sites can be added anywhere throughoutthe amino acid sequence of the optimized binuclease fusion protein.Preferably, the glycosylation sites are introduced in position in theamino acid sequence that does not substantially reduce the nuclease(e.g., RNase and/or DNase) activity of the optimized binuclease fusionprotein.

The addition of O-linked glycosylation sites has been reported to alterserum half-life of proteins, such as growth hormone,follicle-stimulating hormone, IGFBP-6, Factor IX, and many others (e.g.,as disclosed in Okada et al., Endocr Rev 2011; 32:2-342; Weenen et al.,J Clin Endocrinol Metab 2004; 89:5204-12; Marinaro et al., EuropeanJournal of Endocrinology 2000; 142:512-6; US 2011/0154516). Accordingly,in some embodiments, O-linked glycosylation (on serine/threonineresidues) of the optimized binuclease fusion proteins is altered.Methods for altering O-linked glycosylation are routine in the art andcan be achieved, e.g., by beta-elimination (see, e.g., Huang et al.,Rapid Communications in Mass Spectrometry 2002; 16:1199-204; Conrad,Curr Protoc Mol Biol 2001; Chapter 17:Unit17.15A; Fukuda, Curr ProtocMol Biol 2001; Chapter 17; Unit 17.15B; Zachara et al., Curr Protoc MolBiol 2011; Unit 17.6; ); by using commercially available kits (e.g.,GlycoProfile™ Beta-Elimination Kit, Sigma); or by subjecting optimizedbinuclease fusion protein to treatment with a series of exoglycosidasessuch as, but not limited to, β1-4 galactosidase andβ-N-acetylglucosaminidase, until only Gal β1-3GalNAc and/or GlcNAcβ1-3GalNAc remains, followed by treatment with, e.g.,endo-α-N-acetylgalactosaminidase (i.e., O-glycosidase). Such enzymes arecommercially available from, e.g., New England Biolabs. In yet otherembodiments, the optimized binuclease fusion proteins are altered tointroduce O-linked glycosylation in the optimized binuclease fusionprotein as disclosed in, e.g., Okada et al. (supra), Weenen et al.(supra), US2008/0274958; and US2011/0171218. In some embodiments, one ormore O-linked glycosylation consensus sites are introduced into theoptimized binuclease fusion protein, such as CXXGGT/S-C(SEQ ID NO: 33)(van den Steen et al., In Critical Reviews in Biochemistry and MolecularBiology, Michael Cox, ed., 1998; 33:151-208), NST-E/D-A (SEQ ID NO: 34),NITQS (SEQ ID NO: 35), QSTQS (SEQ ID NO: 36), D/E-FT-R/K-V (SEQ ID NO:37), C-E/D-SN (SEQ ID NO: 38), and GGSC-K/R (SEQ ID NO: 39). AdditionalO-linked glycosylation sites can be added anywhere throughout the aminoacid sequence of the optimized binuclease fusion protein. Preferably,the glycosylation sites are introduced in position in the amino acidsequence that does not substantially reduce the nuclease (e.g., RNaseand/or DNase) activity of the optimized binuclease fusion protein.Alternatively, O-linked sugar moieties are introduced by chemicallymodifying an amino acid in the optimized binuclease fusion protein asdescribed in, e.g., WO 87/05330 and Aplin et al., CRC Crit Rev Biochem1981; 259-306).

In some embodiments, both N-linked and O-linked glycosylation sites areintroduced into the optimized binuclease fusion proteins, preferably inpositions in the amino acid sequence that do not substantially reducethe nuclease (e.g., RNase and/or DNase) activity of the optimizedbinuclease fusion protein.

It is well within the abilities of the skilled artisan to introduce,reduce, or eliminate glycosylation (e.g., N-linked or O-linkedglycosylation) in an optimized binuclease fusion protein and determineusing routine methods in the art whether such modifications inglycosylation status increases or decreases the nuclease activity orserum half-life of the optimized binuclease fusion protein.

In some embodiments, the optimized binuclease fusion protein maycomprise an altered glycoform (e.g., an underfucosylated or fucose-freeglycan).

In some embodiments, an optimized binuclease fusion protein with alteredglycosylation has a serum half-life that is increased at least about1.5-fold, such as at least 3-fold, at least 5-fold, at least 10-fold, atleast about 20-fold, at least about 50-fold, at least about 100-fold, atleast about 200-fold, at least about 300-fold, at least about 400-fold,at least about 500-fold, at least about 600-fold, at least about700-fold, at least about 800-fold, at least about 900-fold, at leastabout 1000-fold, or 1000-fold or greater relative to the correspondingglycosylated optimized binuclease fusion proteins (e.g., an optimizedbinuclease fusion protein in which potential N-linked glycosylationsites are not mutated). Routine art-recognized methods can be used todetermine the serum half-life of optimized binuclease fusion proteinswith altered glycosylation status.

In some embodiments, an optimized binuclease fusion protein with alteredglycosylation (e.g., a aglycosylated, deglycosylated, orunderglycosylated optimized binuclease fusion proteins) retains at least50%, such as at least 60%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, at least 99.5%, or 100% of the activity of the correspondingglycosylated optimized binuclease fusion protein (e.g., an optimizedbinuclease fusion protein in which potential N-linked glycosylationsites are not mutated).

In some embodiments, altering the glycosylation status of the optimizedbinuclease fusion proteins may increase nuclease activity, either bydirectly increasing enzymatic activity, or by increasing bioavailability(e.g., serum half-life). Accordingly, in some embodiments, the nucleaseactivity of an optimized binuclease fusion protein with alteredglycosylation is increased by at least 1.3-fold, such as at least1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least5.5-fold, at least 6-fold, at least 6.5-fold, at least 7-fold, at least7.5-fold, at least 8-fold, at least 8.5-fold, at least 9-fold, at least9.5 fold, or 10-fold or greater, relative to the correspondingglycosylated optimized binuclease fusion protein (e.g., an optimizedbinuclease fusion protein in which potential N-linked glycosylationsites are not mutated).

The skilled artisan can readily determine the glycosylation status ofoptimized binuclease fusion proteins using art-recognized methods. In apreferred embodiment, the glycosylation status is determined using massspectrometry. In other embodiments, interactions with Concanavalin A(Con A) can be assessed to determine whether an optimized binucleasefusion protein is underglycosylated. An underglycosylated optimizedbinuclease fusion protein is expected to exhibit reduced binding to ConA-Sepharose when compared to the corresponding glycosylated optimizedbinuclease fusion protein. SDS-PAGE analysis can also be used to comparethe mobility of an underglycosylated protein and correspondingglycosylated protein. The underglycosylated protein is expected to havea greater mobility in SDS-PAGE compared to the glycosylated protein.Other suitable art-recognized methods for analyzing proteinglycosylation status are disclosed in, e.g., Roth et al., InternationalJournal of Carbohydrate Chemistry 2012; 1-10.

Pharmacokinetics, such as serum half-life, of optimized binucleasefusion proteins with different glycosylation status can be assayed usingroutine methods, e.g., by introducing the optimized binuclease fusionproteins in mice, e.g., intravenously, taking blood samples atpre-determined time points, and assaying and comparing levels and/orenzymatic activity of the optimized binuclease fusion proteins in thesamples.

Pharmaceutical Compositions

In certain embodiments, an optimized binuclease fusion protein isadministered alone. In certain embodiments, an optimized binucleasefusion protein is administered prior to the administration of at leastone other therapeutic agent. In certain embodiments, an optimizedbinuclease fusion protein is administered concurrent with theadministration of at least one other therapeutic agent. In certainembodiments, an optimized binuclease fusion protein is administeredsubsequent to the administration of at least one other therapeuticagent. In other embodiments, an optimized binuclease fusion protein isadministered prior to the administration of at least one othertherapeutic agent. As will be appreciated by one of skill in the art, insome embodiments, the optimized binuclease fusion protein is combinedwith the other agent/compound. In some embodiments, the optimizedbinuclease fusion protein and other agent are administered concurrently.In some embodiments, the optimized binuclease fusion protein and otheragent are not administered simultaneously, with the optimized binucleasefusion protein being administered before or after the agent isadministered. In some embodiments, the subject receives both theoptimized binuclease fusion protein and the other agent during a sameperiod of prevention, occurrence of a disorder, and/or period oftreatment.

Pharmaceutical compositions of the invention can be administered incombination therapy, i.e., combined with other agents. In certainembodiments, the combination therapy comprises the optimized binucleasefusion protein, in combination with at least one other agent. Agentsinclude, but are not limited to, in vitro synthetically preparedchemical compositions, antibodies, antigen binding regions, andcombinations and conjugates thereof. In certain embodiments, an agentcan act as an agonist, antagonist, allosteric modulator, or toxin.

In certain embodiments, the invention provides for pharmaceuticalcompositions comprising a optimized binuclease fusion protein togetherwith a pharmaceutically acceptable diluent, carrier, solubilizer,emulsifier, preservative and/or adjuvant.

In certain embodiments, the invention provides for pharmaceuticalcompositions comprising a optimized binuclease fusion protein and atherapeutically effective amount of at least one additional therapeuticagent, together with a pharmaceutically acceptable diluent, carrier,solubilizer, emulsifier, preservative and/or adjuvant.

In certain embodiments, acceptable formulation materials preferably arenontoxic to recipients at the dosages and concentrations employed. Insome embodiments, the formulation material(s) are for s.c. and/or I.V.administration. In certain embodiments, the pharmaceutical compositioncan contain formulation materials for modifying, maintaining orpreserving, for example, the pH, osmolality, viscosity, clarity, color,isotonicity, odor, sterility, stability, rate of dissolution or release,adsorption or penetration of the composition. In certain embodiments,suitable formulation materials include, but are not limited to, aminoacids (such as glycine, glutamine, asparagine, arginine or lysine);antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite orsodium hydrogen-sulfite); buffers (such as borate, bicarbonate,Tris-HCl, citrates, phosphates or other organic acids); bulking agents(such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine,polyvinylpyrrolidone, beta-cyclodextrin orhydroxypropyl-beta-cyclodextrin); fillers; monosaccharides;disaccharides; and other carbohydrates (such as glucose, mannose ordextrins); proteins (such as gelatin); coloring, flavoring and dilutingagents; emulsifying agents; hydrophilic polymers (such aspolyvinylpyrrolidone); low molecular weight polypeptides; salt-formingcounterions (such as sodium); preservatives (such as benzalkoniumchloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol,methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogenperoxide); solvents (such as glycerin, propylene glycol or polyethyleneglycol); sugar alcohols (such as mannitol or sorbitol); suspendingagents; surfactants or wetting agents (such as pluronics, PEG, sorbitanesters, polysorbates such as polysorbate 20, polysorbate 80, triton,tromethamine, lecithin, cholesterol, tyloxapal); stability enhancingagents (such as sucrose or sorbitol); tonicity enhancing agents (such asalkali metal halides, preferably sodium or potassium chloride, mannitolsorbitol); delivery vehicles; diluents; excipients and/or pharmaceuticaladjuvants. (Remington's Pharmaceutical Sciences, 18th Edition, A. R.Gennaro, ed., Mack Publishing Company (1995). In some embodiments, theformulation comprises PBS; 20 mM NaOAC, pH 5.2, 50 mM NaCl; and/or 10 mMNAOAC, pH 5.2, 9% Sucrose.

In certain embodiments, a optimized binuclease fusion protein and/or atherapeutic molecule is linked to a half-life extending vehicle known inthe art. Such vehicles include, but are not limited to, polyethyleneglycol, glycogen (e.g., glycosylation of the optimized binuclease fusionprotein), and dextran. Such vehicles are described, e.g., in U.S.application Ser. No. 09/428,082, now U.S. Pat. No. 6,660,843 andpublished PCT Application No. WO 99/25044.

In certain embodiments, the optimal pharmaceutical composition will bedetermined by one skilled in the art depending upon, for example, theintended route of administration, delivery format and desired dosage.See, for example, Remington's Pharmaceutical Sciences, supra. In certainembodiments, such compositions may influence the physical state,stability, rate of in vivo release and rate of in vivo clearance of theantibodies of the invention.

In certain embodiments, the primary vehicle or carrier in apharmaceutical composition can be either aqueous or non-aqueous innature. For example, in certain embodiments, a suitable vehicle orcarrier can be water for injection, physiological saline solution orartificial cerebrospinal fluid, possibly supplemented with othermaterials common in compositions for parenteral administration. In someembodiments, the saline comprises isotonic phosphate-buffered saline. Incertain embodiments, pharmaceutical compositions comprise Tris buffer ofabout pH 7.0-8.5, or acetate buffer of about H 4.0-5.5, which canfurther include sorbitol or a suitable substitute therefore. In certainembodiments, a composition comprising an optimized binuclease fusionprotein, with or without at least one additional therapeutic agents, canbe prepared for storage by mixing the selected composition having thedesired degree of purity with optional formulation agents (Remington'sPharmaceutical Sciences, supra) in the form of a lyophilized cake or anaqueous solution. Further, in certain embodiments, a compositioncomprising an optimized binuclease fusion protein, with or without atleast one additional therapeutic agent, can be formulated as alyophilizate using appropriate excipients such as sucrose.

In certain embodiments, the pharmaceutical composition can be selectedfor parenteral delivery. In certain embodiments, the compositions can beselected for inhalation or for delivery through the digestive tract,such as orally. The preparation of such pharmaceutically acceptablecompositions is within the ability of one skilled in the art.

In certain embodiments, the formulation components are present inconcentrations that are acceptable to the site of administration. Incertain embodiments, buffers are used to maintain the composition atphysiological pH or at a slightly lower pH, typically within a pH rangeof from about 5 to about 8.

In certain embodiments, when parenteral administration is contemplated,a therapeutic composition can be in the form of a pyrogen-free,parenterally acceptable aqueous solution comprising a desired optimizedbinuclease fusion protein, with or without additional therapeuticagents, in a pharmaceutically acceptable vehicle. In certainembodiments, a vehicle for parenteral injection is sterile distilledwater in which an optimized binuclease fusion protein, with or withoutat least one additional therapeutic agent, is formulated as a sterile,isotonic solution, properly preserved. In certain embodiments, thepreparation can involve the formulation of the desired molecule with anagent, such as injectable microspheres, bio-erodible particles,polymeric compounds (such as polylactic acid or polyglycolic acid),beads or liposomes, that can provide for the controlled or sustainedrelease of the product which can then be delivered via a depotinjection. In certain embodiments, hyaluronic acid can also be used, andcan have the effect of promoting sustained duration in the circulation.In certain embodiments, implantable drug delivery devices can be used tointroduce the desired molecule.

In certain embodiments, a pharmaceutical composition can be formulatedfor inhalation. In certain embodiments, an optimized binuclease fusionprotein, with or without at least one additional therapeutic agent, canbe formulated as a dry powder for inhalation. In certain embodiments, aninhalation solution comprising an optimized binuclease fusion protein,with or without at least one additional therapeutic agent, can beformulated with a propellant for aerosol delivery. In certainembodiments, solutions can be nebulized. Pulmonary administration isfurther described in PCT application no. PCT/US94/001875, whichdescribes pulmonary delivery of chemically modified proteins.

In certain embodiments, it is contemplated that formulations can beadministered orally. In certain embodiments, an optimized binucleasefusion protein, with or without at least one additional therapeuticagents, that is administered in this fashion can be formulated with orwithout those carriers customarily used in the compounding of soliddosage forms such as tablets and capsules. In certain embodiments, acapsule can be designed to release the active portion of the formulationat the point in the gastrointestinal tract when bioavailability ismaximized and pre-systemic degradation is minimized. In certainembodiments, at least one additional agent can be included to facilitateabsorption of an optimized binuclease fusion protein and/or anyadditional therapeutic agents. In certain embodiments, diluents,flavorings, low melting point waxes, vegetable oils, lubricants,suspending agents, tablet disintegrating agents, and binders can also beemployed.

In certain embodiments, a pharmaceutical composition can involve aneffective quantity of an optimized binuclease fusion protein, with orwithout at least one additional therapeutic agents, in a mixture withnon-toxic excipients which are suitable for the manufacture of tablets.In certain embodiments, by dissolving the tablets in sterile water, oranother appropriate vehicle, solutions can be prepared in unit-doseform. In certain embodiments, suitable excipients include, but are notlimited to, inert diluents, such as calcium carbonate, sodium carbonateor bicarbonate, lactose, or calcium phosphate; or binding agents, suchas starch, gelatin, or acacia; or lubricating agents such as magnesiumstearate, stearic acid, or talc.

Additional pharmaceutical compositions will be evident to those skilledin the art, including formulations involving an optimized binucleasefusion protein, with or without at least one additional therapeuticagent(s), in sustained- or controlled-delivery formulations. In certainembodiments, techniques for formulating a variety of other sustained- orcontrolled-delivery means, such as liposome carriers, bio-erodiblemicroparticles or porous beads and depot injections, are also known tothose skilled in the art. See for example, PCT Application No.PCT/US93/00829 which describes the controlled release of porouspolymeric microparticles for the delivery of pharmaceuticalcompositions. In certain embodiments, sustained-release preparations caninclude semipermeable polymer matrices in the form of shaped articles,e.g. films, or microcapsules. Sustained release matrices can includepolyesters, hydrogels, polylactides (U.S. Pat. No. 3,773,919 and EP058,481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate(Sidman et al, Biopolymers, 22:547-556 (1983)), poly(2-hydroxyethyl-methacrylate) (Langer et al., J Biomed Mater Res, 15:167-277 (1981) and Langer, Chem Tech, 12:98-105 (1982)), ethylene vinylacetate (Langer et al, supra) or poly-D(−)-3-hydroxybutyric acid (EP133,988). In certain embodiments, sustained release compositions canalso include liposomes, which can be prepared by any of several methodsknown in the art. See, e.g., Eppstein et al, PNAS, 82:3688-3692 (1985);EP 036,676; EP 088,046 and EP 143,949.

The pharmaceutical composition to be used for in vivo administrationtypically is sterile. In certain embodiments, this can be accomplishedby filtration through sterile filtration membranes. In certainembodiments, where the composition is lyophilized, sterilization usingthis method can be conducted either prior to or following lyophilizationand reconstitution. In certain embodiments, the composition forparenteral administration can be stored in lyophilized form or in asolution. In certain embodiments, parenteral compositions generally areplaced into a container having a sterile access port, for example, anintravenous solution bag or vial having a stopper pierceable by ahypodermic injection needle.

In certain embodiments, once the pharmaceutical composition has beenformulated, it can be stored in sterile vials as a solution, suspension,gel, emulsion, solid, or as a dehydrated or lyophilized powder. Incertain embodiments, such formulations can be stored either in aready-to-use form or in a form (e.g., lyophilized) that is reconstitutedprior to administration.

In certain embodiments, kits are provided for producing a single-doseadministration unit. In certain embodiments, the kit can contain both afirst container having a dried protein and a second container having anaqueous formulation. In certain embodiments, kits containing single andmulti-chambered pre-filled syringes (e.g., liquid syringes andlyosyringes) are included.

In certain embodiments, the effective amount of a pharmaceuticalcomposition comprising an optimized binuclease fusion protein, with orwithout at least one additional therapeutic agent, to be employedtherapeutically will depend, for example, upon the therapeutic contextand objectives. One skilled in the art will appreciate that theappropriate dosage levels for treatment, according to certainembodiments, will thus vary depending, in part, upon the moleculedelivered, the indication for which an optimized binuclease fusionprotein, with or without at least one additional therapeutic agent, isbeing used, the route of administration, and the size (body weight, bodysurface or organ size) and/or condition (the age and general health) ofthe patient. In certain embodiments, the clinician can titer the dosageand modify the route of administration to obtain the optimal therapeuticeffect. In certain embodiments, a typical dosage can range from about0.1 μg/kg to up to about 100 mg/kg or more, depending on the factorsmentioned above. In certain embodiments, the dosage can range from 0.1μg/kg up to about 100 mg/kg; or 1 μg/kg up to about 100 mg/kg; or 5μg/kg up to about 100 mg/kg.

In certain embodiments, the frequency of dosing will take into accountthe pharmacokinetic parameters of an optimized binuclease fusion proteinand/or any additional therapeutic agents in the formulation used. Incertain embodiments, a clinician will administer the composition until adosage is reached that achieves the desired effect. In certainembodiments, the composition can therefore be administered as a singledose, or as two or more doses (which may or may not contain the sameamount of the desired molecule) over time, or as a continuous infusionvia an implantation device or catheter. Further refinement of theappropriate dosage is routinely made by those of ordinary skill in theart and is within the ambit of tasks routinely performed by them. Incertain embodiments, appropriate dosages can be ascertained through useof appropriate dose-response data.

In certain embodiments, the route of administration of thepharmaceutical composition is in accord with known methods, e.g. orally,through injection by intravenous, intraperitoneal, intracerebral(intra-parenchymal), intracerebroventricular, intramuscular,subcutaneously, intra-ocular, intraarterial, intraportal, orintralesional routes; by sustained release systems or by implantationdevices. In certain embodiments, the compositions can be administered bybolus injection or continuously by infusion, or by implantation device.

In certain embodiments, the composition can be administered locally viaimplantation of a membrane, sponge or another appropriate material ontowhich the desired molecule has been absorbed or encapsulated. In certainembodiments, where an implantation device is used, the device can beimplanted into any suitable tissue or organ, and delivery of the desiredmolecule can be via diffusion, timed-release bolus, or continuousadministration.

In certain embodiments, it can be desirable to use a pharmaceuticalcomposition comprising an optimized binuclease fusion protein, with orwithout at least one additional therapeutic agent, in an ex vivo manner.In such instances, cells, tissues and/or organs that have been removedfrom the patient are exposed to a pharmaceutical composition comprisingan optimized binuclease fusion protein, with or without at least oneadditional therapeutic agent, after which the cells, tissues and/ororgans are subsequently implanted back into the patient.

In certain embodiments, an optimized binuclease fusion protein and/orany additional therapeutic agents can be delivered by implanting certaincells that have been genetically engineered, using methods such as thosedescribed herein, to express and secrete the polypeptides. In certainembodiments, such cells can be animal or human cells, and can beautologous, heterologous, or xenogeneic. In certain embodiments, thecells can be immortalized. In certain embodiments, in order to decreasethe chance of an immunological response, the cells can be encapsulatedto avoid infiltration of surrounding tissues. In certain embodiments,the encapsulation materials are typically biocompatible, semi-permeablepolymeric enclosures or membranes that allow the release of the proteinproduct(s) but prevent the destruction of the cells by the patient'simmune system or by other detrimental factors from the surroundingtissues.

In Vitro Assays

Various in vitro assays known in the art can be used to assess theefficacy of the optimized binuclease fusion proteins of the invention.

For example, cultured human PBMCs from normal or lupus patient PBMCs areisolated, cultured, and treated with various stimuli (e.g., TLR ligands,costimulatory antibodies, immune complexes, and normal or autoimmunesera), in the presence or absence of the optimized binuclease fusionproteins. Cytokine production by the stimulated cells can be measuredusing commercially available reagents, such as the antibody pair kitsfrom Biolegend (San Diego, Calif.) for various cytokines (e.g., IL-6,IL-8, IL-10, IL-4, IFN-gamma, and TNF-alpha). Culture supernatants areharvested at various time points as appropriate for the assay (e.g., 24,48 hours, or later time points) to determine the effects that theoptimized binuclease fusion proteins have on cytokine production.IFN-alpha production is measured using, e.g., anti-human IFN-alphaantibodies and standard curve reagents available from PBL interferonsource (Piscataway, N.J.). Similar assays are performed using humanlymphocyte subpopulations (isolated monocytes, B cells, pDCs, T cells,etc.); purified using, e.g., commercially available magnetic bead basedisolation kits available from Miltenyi Biotech (Auburn, Calif.).

Multi-color flow cytometry can be used to assess the effects of theoptimized binuclease fusion proteins on immune cell activation bymeasuring the expression of lymphocyte activation receptors such as CD5,CD23, CD69, CD80, CD86, and CD25 in PBMCs or isolated cellsubpopulations at various time points after stimulation using routineart-recognized methods.

The efficacy of optimized binuclease fusion proteins can also be testedby incubating SLE patient serum with normal human pDCs to activate IFNoutput, as described in, e.g., Ahlin et al., Lupus 2012:21:586-95;Mathsson et al., Clin Expt Immunol 2007; 147:513-20; and Chiang et al.,JImmunol 2011; 186:1279-1288. Without being bound by theory, circulatingnucleic acid-containing immune complexes in SLE patient sera facilitatenucleic acid antigen entry into pDC endosomes via Fc receptor-mediatedendocytosis, followed by binding of nucleic acids to and activation ofendosomal TLRs 7, 8, and 9. To assess the impact of the optimizedbinuclease fusion proteinss, SLE patient sera or plasma is pretreatedwith the optimized binuclease fusion proteins, followed by addition tocultures of pDC cells isolated from healthy volunteers. Levels of IFN-αproduced are then determined at multiple time points. By degradingnucleic-acid containing immune complexes, effective optimized binucleasefusion proteins are expected to reduce the quantity of IFN-α produced.

The effectiveness of optimized binuclease fusion proteins isdemonstrated by comparing the results of an assay from cells treatedwith an optimized binuclease fusion protein disclosed herein to theresults of the assay from cells treated with control formulations. Aftertreatment, the levels of the various markers (e.g., cytokines,cell-surface receptors, proliferation) described above are generallyimproved in an effective optimized binuclease fusion protein treatedgroup relative to the marker levels existing prior to the treatment, orrelative to the levels measured in a control group.

Methods of Treatment

The optimized binuclease fusion proteins of the disclosure areparticularly effective in the treatment of autoimmune disorders orabnormal immune responses. In this regard, it will be appreciated thatthe optimized binuclease fusion proteins of the present disclosure maybe used to control, suppress, modulate, treat, or eliminate unwantedimmune responses to both external and autoantigens.

In another aspect, an optimized binuclease fusion protein is adapted forpreventing (prophylactic) or treating (therapeutic) a disease ordisorder, such as an autoimmune disease, in a mammal by administering anoptimized binuclease fusion protein in a therapeutically effectiveamount or a sufficient amount to the mammal in need thereof, wherein thedisease is prevented or treated. Any route of administration suitablefor achieving the desired effect is contemplated by the invention (e.g.,intravenous, intramuscular, subcutaneous). Treatment of the diseasecondition may result in a decrease in the symptoms associated with thecondition, which may be long-term or short-term, or even a transientbeneficial effect.

Numerous disease conditions are suitable for treatment with optimizedbinuclease fusion proteins of the disclosure. For example, in someaspects, the disease or disorder is an autoimmune disease or cancer. Insome such aspects, the autoimmune disease is insulin-dependent diabetesmellitus, multiple sclerosis, experimental autoimmune encephalomyelitis,rheumatoid arthritis, experimental autoimmune arthritis, myastheniagravis, thyroiditis, an experimental form of uveoretinitis, Hashimoto'sthyroiditis, primary myxoedema, thyrotoxicosis, pernicious anaemia,autoimmune atrophic gastritis, Addison's disease, premature menopause,male infertility, juvenile diabetes, Goodpasture's syndrome, pemphigusvulgaris, pemphigoid, sympathetic ophthalmia, phacogenic uveitis,autoimmune haemolytic anaemia, idiopathic leucopenia, primary biliarycirrhosis, active chronic hepatitis Hbs-ve, cryptogenic cirrhosis,ulcerative colitis, Sjogren's syndrome, scleroderma, Wegener'sgranulomatosis, polymyositis, dermatomyositis, discoid LE, SLE, orconnective tissue disease.

In a specific embodiment, an optimized binuclease fusion protein is usedto prevent or treat SLE or Sjogren's syndrome. The effectiveness of anoptimized binuclease fusion protein is demonstrated by comparing theIFN-alpha levels, IFN-alpha response gene levels, autoantibody titers,kidney function and pathology, and/or circulating immune complex levelsin mammals treated with an optimized binuclease fusion protein disclosedherein to mammals treated with control formulations.

For example, a human subject in need of treatment is selected oridentified (e.g., a patient who fulfills the American College ofRheumatology criteria for SLE, or a patient who fulfills theAmerican-European Consensus Sjogren's Classification Criteria). Thesubject can be in need of, e.g., reducing a cause or symptom of SLE orSjogren's syndrome. The identification of the subject can occur in aclinical setting, or elsewhere, e.g., in the subject's home through thesubject's own use of a self-testing kit.

At time zero, a suitable first dose of an optimized binuclease fusionprotein is administered to the subject. The optimized binuclease fusionprotein is formulated as described herein. After a period of timefollowing the first dose, e.g., 7 days, 14 days, and 21 days, thesubject's condition is evaluated, e.g., by measuring IFN-alpha levels,IFN-alpha response gene levels, autoantibody titers, kidney function andpathology, and/or circulating immune complex levels. Other relevantcriteria can also be measured. The number and strength of doses areadjusted according to the subject's needs. After treatment, thesubject's IFN-alpha levels, IFN-alpha response gene levels, autoantibodytiters, kidney function and pathology, and/or circulating immune complexlevels are lowered and/or improved relative to the levels existing priorto the treatment, or relative to the levels measured in a similarlyafflicted but untreated/control subject.

In another example, a rodent subject in need of treatment is selected oridentified (see, e.g., Example 7). The identification of the subject canoccur in a laboratory setting or elsewhere. At time zero, a suitablefirst dose of an optimized binuclease fusion protein is administered tothe subject. The optimized binuclease fusion protein is formulated asdescribed herein. After a period of time following the first dose, e.g.,7 days, 14 days, and 21 days, the subject's condition is evaluated,e.g., by measuring IFN-alpha levels, IFN-alpha response gene levels,autoantibody titers, kidney function and pathology, and/or circulatingimmune complex levels. Other relevant criteria can also be measured. Thenumber and strength of doses are adjusted according to the subject'sneeds.

After treatment, the subject's IFN-alpha levels, IFN-alpha response genelevels, autoantibody titers, kidney function and pathology, and/orcirculating immune complex levels are lowered and/or improved relativeto the levels existing prior to the treatment, or relative to the levelsmeasured in a similarly afflicted but untreated/control subject.

Another aspect of the present invention is to use gene therapy methodsfor treating or preventing disorders, diseases, and conditions with oneor more optimized binuclease fusion proteins. The gene therapy methodsrelate to the introduction of optimized binuclease fusion proteinnucleic acid (DNA, RNA and antisense DNA or RNA) sequences into ananimal in need thereof to achieve expression of the polypeptide orpolypeptides of the present disclosure. This method can includeintroduction of one or more polynucleotides encoding an optimizedbinuclease fusion protein of the present disclosure operably coupled toa promoter and any other genetic elements necessary for the expressionof the polypeptide by the target tissue.

In gene therapy applications, optimized binuclease fusion protein genesare introduced into cells in order to achieve in vivo synthesis of atherapeutically effective genetic product. “Gene therapy” includes bothconventional gene therapies where a lasting effect is achieved by asingle treatment, and the administration of gene therapeutic agents,which involves the one time or repeated administration of atherapeutically effective DNA or mRNA. The oligonucleotides can bemodified to enhance their uptake, e.g., by substituting their negativelycharged phosphodiester groups by uncharged groups.

EXAMPLES

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for.

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of protein chemistry, biochemistry,recombinant DNA techniques and pharmacology, within the skill of theart. Such techniques are explained fully in the literature. See, e.g.,T. E. Creighton, Proteins: Structures and Molecular Properties (W.H.Freeman and Company, 1993); A. L. Lehninger, Biochemistry (WorthPublishers, Inc., current addition); Sambrook, et al, Molecular Cloning:A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S.Colowick and N. Kaplan eds., Academic Press, Inc.); Remington'sPharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack PublishingCompany, 1990); Carey and Sundberg Advanced Organic Chemistry 3^(rd) Ed.(Plenum Press) Vols A and B (1992).

Example 1 Generating Optimized Binuclease Fusion Protein EncodingExpression Vectors

Various embodiments of the optimized binuclease fusion proteins of thedisclosure are shown in FIG. 1, with amino acid sequences of eachpresented in the Sequence Table. As exemplary optimized binucleasefusion proteins, binuclease fusion proteins with the configurationsshown in FIG. 1 were constructed. Specifically, starting from the aminoacid sequence of the optimized binuclease fusion proteins,polynucleotides encoding the optimized binuclease fusion proteins weredirectly synthesized using codon optimization by Genescript (Genescript,Piscatawy, N.J.) to allow for optimal expression in mammalian cells. Theprocess of optimization involved, e.g., avoiding regions of very high(>80%) or very low (<30%) GC content when possible, and avoidingcis-acting sequence motifs, such as internal TATA-boxes, chi-sites andribosomal entry sites, AT-rich or GC-rich sequence stretches, RNAinstability motifs, repeat sequences and RNA secondary structures, andcryptic splice donor and acceptor sites in higher eukaryotes. DNAsencoding the optimized binuclease fusion proteins are cloned into thepcDNA3.1+mammalian expression vector. Optimized binuclease fusionproteins with the following configurations were generated.

Tandem homodimer RSLV-145 (SEQ ID NO: 1) has the configurationDNase-linker-RNase-Fc, wherein a wild-type, human RNase1 domain (SEQ IDNO: 27) is operably coupled without a linker to the N-terminus to amutant Fc region comprising SCC hinge and CH2 mutations P238S, P331S(SEQ ID NO: 55) and a mutant human DNase1 domain (SEQ ID NO: 25) isoperably coupled to the N-terminus of the RNase1 domain via a NLG linker(SEQ ID NO: 41).

To preferentially form heterodimers, each of the Fc domains in thefollowing constructs included complementary CH3 mutations: T350V, L351Y,F405A, and Y407V; and T350V, T366L, K392L, and T394W (numberingaccording to the EU index.)

Tandem heterodimer RSLV-147 has the configuration DNase-Fc (SEQ ID NO:3) and RNase-Fc (SEQ ID NO: 4), wherein a mutant human DNase1 domain(SEQ ID NO: 25) is operably coupled to the N-terminus of a first mutantFc region comprising SCC hinge, CH2 mutations P238S, P331S and CH3mutations, and wherein a wild-type, human RNase1 domain (SEQ ID NO: 27)is operably coupled to the N-terminus of a second mutant Fc regioncomprising SCC hinge, CH2 mutations P238S, P331S and CH3 mutations.

Heterodimer RSLV-148 has the configuration DNase-first Fcdomain-linker-RNase (SEQ ID NO: 5) and a mutant second Fc domain(N-terminal truncation including first cysteine in CCC hinge) comprisingCH2 mutations P238S, P331S and CH3 mutations (SEQ ID NO: 6), wherein amutant human DNase1 domain (SEQ ID NO: 25) is operably coupled to theN-terminus of a first mutant Fc region comprising SCC hinge, CH2mutations P238S, P331S and CH3 mutations, and wherein a wild-type, humanRNase1 domain (SEQ ID NO: 27) is operably coupled via an NLG linker tothe C-terminus of the first Fc region.

Heterodimer RSLV-149 has the configuration DNase-Fc (SEQ ID NO: 7) andFc-linker-RNase (SEQ ID NO: 8), wherein a mutant human DNase1 domain(SEQ ID NO: 25) is operably coupled to the N-terminus of a first mutantFc region comprising SCC hinge, CH2 mutations P238S, P331S and CH3mutations, and wherein a wild-type, human RNase1 domain (SEQ ID NO: 27)is operably coupled via an NLG linker to the C-terminus of a secondmutant Fc region comprising CH2 mutations P238S, P331S and CH3 mutations(SEQ ID NO:6).

Heterodimer RSLV-152 has the configuration RNase-first mutantFc-linker-DNase (SEQ ID NO: 13) and a second mutant Fc domain comprisingCH2 mutations P238S, P331S, and CH3 mutations (SEQ ID NO: 14), wherein awild-type, human RNase1 domain (SEQ ID NO: 27) is operably coupled tothe N-terminus of a first mutant Fc region comprising SCC hinge and CH2mutations P238S, P331S and CH3 mutations, and wherein a mutant humanDNase1 domain (SEQ ID NO: 25) is operably coupled via an NLG liner tothe C-terminus of the first mutant Fc region.

Heterodimer RSLV-153 has the configuration Fc-linker-DNase (SEQ ID NO:15) and RNase-Fc (SEQ ID NO: 16), wherein a mutant human DNase1 domain(SEQ ID NO: 25) is operably coupled via an NLG linker to the C-terminusof a first mutant Fc region comprising CH2 mutations P238S, P331S andCH3 mutations, and wherein a wild-type, human RNase1 domain (SEQ ID NO:27) is operably coupled to the N-terminus of a second mutant Fc regioncomprising SCC hinge, CH2 mutations P238S, P331S and CH3 mutations.

Constructs RLSV-327 (a binuclease containing RNase 1 and DNase 1 linkedto human serum albumin; RNase-linker-HSA-linker-DNaseE13R/N74K/A114F/T205K) and RSLV-132 (RNase-Fc), containing DNase andRNase moieties, were used as controls.

Example 2 Transient Expression of and Stable Mammalian Cell LinesExpressing Optimized Binuclease Fusion Proteins

For transient expression, expression vectors from Example 1 containingthe optimized binuclease fusion protein inserts are transientlytrasfected using FreeStyle™ MAX Reagent into Chinese Hamster Ovary (CHO)cells, e.g., CHO-S cells (e.g., FreeStyle™ CHO-S cells, Invitrogen),using the manufacturer recommended transfection protocol. CHO-S cellsare maintained in FreeStyle™ CHO Expression Medium containing 2 mML-Glutamine and penicillin-streptomycin.

Stable CHO-S cell lines expressing the optimized binuclease fusionproteins are generated using routine methods known in the art. Forexample, CHO-S cells can be infected with a virus (e.g., retrovirus,lentivirus) comprising the nucleic acid sequences of an optimizedbinuclease fusion protein, as well as the nucleic acid sequencesencoding a marker (e.g., GFP, surface markers selectable by magneticbeads) that is selected for using, e.g., flow cytometry or magnetic beadseparation (e.g., MACSelect™ system). Alternatively, CHO-S cells aretransfected using any transfection method known in the art, such aselectroporation (Lonza) or the FreeStyle™ MAX Reagent as mentionedabove, with a vector comprising the nucleic acid sequences of theoptimized binuclease fusion proteins and a selectable marker, followedby selection using, e.g., flow cytometry. The selectable marker can beincorporated into the same vector as that encoding the optimizedbinuclease fusion proteins or a separate vector.

Optimized binuclease fusion proteins are purified from culturesupernatant by capturing the molecules using a column packed withProtein-A sepharose beads, followed by washes in column wash buffer(e.g., 90 mM Tris, 150 mM NaCl, 0.05% sodium azide) and releasing themolecules from the column using a suitable elution buffer (e.g., 0.1 Mcitrate buffer, pH 3.0). The eluted material is further concentrated bybuffer exchange through serial spins in PBS using Centriconconcentrators, followed by filtration through 0.2 m filter devices. Theconcentration of the optimized binuclease fusion proteins is determinedusing standard spectrophotometric methods (e.g., Bradford, BCA, Lowry,Biuret assays).

Example 3 Nuclease Activity of Purified Optimized Binuclease FusionProteins

RNase activity of optimized binuclease fusion proteins present in mousesera was analyzed. The proteins were added at doses of 12.5 to 100 ng to2.5 mg/ml of poly-IC (Sigma) in 50 mM Hepes and 100 mM NaCl at pH 7.3and incubated for 37° C. for 50 minutes. TCA was added to finalconcentration of 5% and left on ice. Samples were filtered to removeprecipated and the filtrate was collected for OD₂₆₀ readings. Theresults are shown in FIG. 2. RSLV-132 and RSLV-145, both containing 2RNase moieties per molar equivalent of construct, were both active, withRSLV-145 being more active than RSLV-132. The other constructs(RSLV-147, RSLV-152, RSLV-153 and RSLV-327), containing only one RNasemoiety on a molar basis, were comparable and in line with RSLV-132.Surprisingly, RSLV-148 and RLSV-149 possessed activity greater thanRSLV-132 and other single RNase constructs. These constructs eachcontain the RNase moiety attached at the C-terminus of the Fc domain.

DNase1 activity of the optimized binuclease fusion proteins containingDNase1 domains was measured using ODN-2006-G5 (InvivoGen, tlrl-2006g5),a DNA oligonucleotide agonist of TLR9. The proteins, in dosages rangingfrom 0.24 ng/ml to 500 ng/ml) were incubated for 1 hour at 37° C. withODN-2006-G5 in DMEM containing 25 mM Hepes and 10% FBS. The reactionmixtures were applied to hTLR9 HEKBlue cells, engineered to secretealkaline phosphatase (SEAP) in response to TLR9 agonists, overnight at37° C. The culture media was harvested and assayed for SEAP using acolormetric substrate and then read at OD₆₂₀. IC50 values werecalculated using GraphPad Prism® version 6.0e software. Results areshown in FIG. 3. All 6 constructs (RSLV-145, RSLV-147, RSLV-148,RSLV-149, RSLV-152 and RSLV-153) possessed robust DNase activity andwere at least 5000-fold more active than recombinant huDNase1. RSLV-145appeared to be approximately 2× more active than other heterodimerconstructs and RSLV-327, likely due to the 2 DNase domains in RSLV-145compared to 1 DNase domain in the other constructs. RSLV-152 wasconsistently the least active of the constructs.

Example 4 Efficacy of Optimized Binuclease Fusion Proteins In VitroEffects of Optimized Binuclease Fusion Proteins on Cytokine Expression

Human PBMCs are isolated from normal patients and lupus patients andcultured. The cells are treated with various stimulatory TLR ligands,costimulatory antibodies, immune complexes, and normal or autoimmunesera, with or without the optimized binuclease fusion proteins ofExample 2. Culture supernatant is collected at various time points(e.g., 6 hrs, 12 hrs, 24 hrs, 48 hrs, etc) and levels of a panel ofcytokines, including human IL-6, IL-8, IL-10, IL-4, IFN-gamma, IFN-alphaand TNF-alpha are measured using commercially available ELISA kits from,e.g., Thermo Fisher Scientific, Inc. Effective optimized binucleasefusion proteins are expected to reduce the levels of cytokines producedby stimulated PBMCs relative to controls.

Effects of Optimized Binuclease Fusion Proteins on Lymphocyte ActivationReceptor Expression

Human PBMCs are isolated from normal patients and lupus patients andcultured. The cells are treated with various stimulatory TLR ligands,costimulatory antibodies, immune complexes, and normal or autoimmunesera, with or without the optimized binuclease fusion proteins ofExample 2. Cells are then subjected to multi-color flow cytometry tomeasure the expression of lymphocyte activation receptors CD5, CD23,CD69, CD80, CD86, and CD25 at various time points (e.g., 6 hrs, 12 hrs,24 hrs, 48 hrs, etc.) after stimulation using routine art-recognizedmethods. Suitable antibodies for these receptors are commerciallyavailable from, e.g., BD/PharMingen. Effective optimized binucleasefusion proteins are expected to reduce the expression of the lymphocyteactivation receptors in stimulated PBMCs relative to controls.

Effects of Optimized Binuclease Fusion Proteins on PlasmacytoidDendritic Cell (pDC) Interferon Output

pDCs from healthy volunteers are isolated using art-recognized methodsor commercially available kits, such as the EasySep™ Human EpCAMPositive Selection Kit (StemCell Technologies, Inc.). Isolated pDCs arecultured in, e.g., 96-well flat-bottom plates, at a densities rangingfrom 5×10⁴ to 2.5×10⁵/well in 0.1 ml in an appropriate medium (e.g.,complete RPMI medium containing 10% FBS, 2 mM glutamine, 55 μMβ-mercaptoethanol, 1 mM sodium pyruvate, 100 U/ml penicillin, and 100μg/ml streptomycin). Cultured pDCs are activated by adding sera orplasma from individual SLE patients diluted with culture medium at a 1:5ratio, and 0.1 ml of these samples are added to the cell-containingwells (final patient serum concentration is 10%). Cultures are incubatedat 37° C. for 40 hr, after which the conditioned media is harvested andassessed for IFNα content using a commercially available ELISA kit.Serum samples obtained from healthy volunteers are used as controls. Toassess the impact of the optimized binuclease fusion proteins, SLEpatient sera or plasma is pretreated with the optimized binucleasefusion proteins (1-10 μg/ml) for 30 min and added to the pDC cultures.Effective optimized binuclease fusion proteins are expected to reducethe quantity of IFNα produced as a result of degrading the nucleicacid-containing ICs.

While the invention has been particularly shown and described withreference to a preferred embodiment and various alternate embodiments,it will be understood by persons skilled in the relevant art thatvarious changes in form and details can be made therein withoutdeparting from the spirit and scope of the invention.

All references, issued patents and patent applications cited within thebody of the instant specification are hereby incorporated by referencein their entirety, for all purposes.

Sequence Table SEQ ID NO Description Sequence  1 RSLV-145 (DNase-LKIAAFNIQTFGRTKMSNATLVSYIVQILSRYDIALVQEV NLG linker-RNase-RDSHLTAVGKLLDNLNQDAPDTYHYVVSEPLGRKSYK Fc) amino acidERYLFVYRPDQVSAVDSYYYDDGCEPCGNDTFNREPFI sequenceVRFFSRFTEVREFAIVPLHAAPGDAVAEIDALYDVYLDV Mature humanQEKWGLEDVMLMGDFNAGCSYVRPSQWSSIRLWTSPT DNase1FQWLIPDSADTTAKPTHCAYDRIVVAGMLLRGAVVPDS E13R/N74K/A114F/ALPFNFQAAYGLSDQLAQAISDHYPVEVMLK VDGASSP T205K (underline) VNVSSPSVQDIKESRAKKFQRQHMDSDSSPSSSSTYCN NLG Linker (bold)QMMRRRNMTQGRCKPVNTFVHEPLVDVQNVCFQEKV Mature humanTCKNGQGNCYKSNSSMHITDCRLTNGSRYPNCAYRTSP RNase1 (boldKERHIIVACEGSPYVPVHFDASVEDSTEPKSSDKTHTCPP underline)CPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVS Fc domainHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGK  2RSLV-146 (Fc- DKTHTCPPCPAPELLGGSSVFLFPPKPKDTLMISRTPEVT linker-RNase-NLGCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY linker-DNase) aminoNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIE acid sequenceKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKG GGGSKESRAKKFQRQHMDSDSSPSSSSTYCNQMMRRR NMTQGRCKPVNTFVHEPLVDVQNVCFQEKVTCKNGQGNCYKSNSSMHITDCRLTNGSRYPNCAYRTSPKERHIIVA CEGSPYVPVHFDASVEDSTVDGASSPVNVSSPSVQDI L KIAAFNIQTFGRTKMSNATLVSYIVQILSRYDIALVQEVRDSHLTAVGKLLDNLNQDAPDTYHYVVSEPLGRKSYKERYLFVYRPDQVSAVDSYYYDDGCEPCGNDTFNREPFIVRFFSRFTEVREFAIVPLHAAPGDAVAEIDALYDVYLDVQEKWGLEDVMLMGDFNAGCSYVRPSQWSSIRLWTSPTFQWLIPDSADTTAKPTHCAYDRIVVAGMLLRGAVVPDS ALPFNFQAAYGLSDQLAQAISDHYPVEVMLK 3 RSLV-147 DNase LKIAAFNIQTFGRTKMSNATLVSYIVQILSRYDIALVQEVchain (Dnase-FcA) RDSHLTAVGKLLDNLNQDAPDTYHYVVSEPLGRKSYKEamino acid sequence RYLFVYRPDQVSAVDSYYYDDGCEPCGNDTFNREPFIVRFFSRFTEVREFAIVPLHAAPGDAVAEIDALYDVYLDVQEKWGLEDVMLMGDFNAGCSYVRPSQWSSIRLWTSPTFQWLIPDSADTTAKPTHCAYDRIVVAGMLLRGAVVPDSALPFNFQAAYGLSDQLAQAISDHYPVEVMLKEPKSSDKTHTCPPCPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYVYPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFALVSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 4 RSLV-147 RNase KESRAKKFQRQHMDSDSSPSSSSTYCNQMMRRRNMTQchain (RNase-FcB) GRCKPVNTFVHEPLVDVQNVCFQEKVTCKNGQGNCYKamino acid sequence SNSSMHITDCRLTNGSRYPNCAYRTSPKERHIIVACEGSPYVPVHFDASVEDSTEPKSSDKTHTCPPCPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYVLPPSRDELTKNQVSLLCLVKGFYPSDIAVEWESNGQPENNYLTWPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK  5RSLV-148 (DNase- LKIAAFNIQTFGRTKMSNATLVSYIVQILSRYDIALVQEVFcA-NLG linker- RDSHLTAVGKLLDNLNQDAPDTYHYVVSEPLGRKSYKE RNase) amino acidRYLFVYRPDQVSAVDSYYYDDGCEPCGNDTFNREPFIV sequenceRFFSRFTEVREFAIVPLHAAPGDAVAEIDALYDVYLDVQEKWGLEDVMLMGDFNAGCSYVRPSQWSSIRLWTSPTFQWLIPDSADTTAKPTHCAYDRIVVAGMLLRGAVVPDSALPFNFQAAYGLSDQLAQAISDHYPVEVMLKEPKSSDKTHTCPPCPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYVYPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFALVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKVDGAS SPVNVSSPSVQDIKESRAKKFQRQHMDSDSSPSSSSTYC NQMMRRRNMTQGRCKPVNTFVHEPLVDVQNVCFQEKVTCKNGQGNCYKSNSSMHITDCRLTNGSRYPNCAYRTS PKERHIIVACEGSPYVPVHFDASVEDST  6RSLV-148 FcB DKTHTCPPCPAPELLGGSSVFLFPPKPKDTLMISRTPEVT chain amino acidCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY sequenceNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYVLPPSRDELTKNQVSLLCLVKGFYPSDIAVEWESNGQPENNYLTWPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK  7 RSLV-149 DNaseLKIAAFNIQTFGRTKMSNATLVSYIVQILSRYDIALVQEV chain (DNase-Fc)RDSHLTAVGKLLDNLNQDAPDTYHYVVSEPLGRKSYKE amino acid sequenceRYLFVYRPDQVSAVDSYYYDDGCEPCGNDTFNREPFIVRFFSRFTEVREFAIVPLHAAPGDAVAEIDALYDVYLDVQEKWGLEDVMLMGDFNAGCSYVRPSQWSSIRLWTSPTFQWLIPDSADTTAKPTHCAYDRIVVAGMLLRGAVVPDSALPFNFQAAYGLSDQLAQAISDHYPVEVMLKEPKSSDKTHTCPPCPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYVYPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFALVSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 8 RSLV-149 RNase DKTHTCPPCPAPELLGGSSVFLFPPKPKDTLMISRTPEVTchain (Fc-NLG- CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY linker-RNase) aminoNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEK acid sequenceTISKAKGQPREPQVYVLPPSRDELTKNQVSLLCLVKGFYPSDIAVEWESNGQPENNYLTWPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKVD GASSPVNVSSPSVQDIKESRAKKFQRQHMDSDSSPSSSS TYCNQMMRRRNMTQGRCKPVNTFVHEPLVDVQNVCFQEKVTCKNGQGNCYKSNSSMHITDCRLTNGSRYPNCAY RTSPKERHIIVACEGSPYVPVHFDASVEDST 9 RSLV-150 DNase EPKSCDKTHTCPPCPAPELLGGSSVFLFPPKPKDTLMISRchain (FcA-NLG TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR linker-DNase) aminoEEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP acid sequenceASIEKTISKAKGQPREPQVYVYPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFALVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GKVDGASSPVNVSSPSVQDILKIAAFNIQTFGRTKMSNA TLVSYIVQILSRYDIALVQEVRDSHLTAVGKLLDNLNQDAPDTYHYVVSEPLGRKSYKERYLFVYRPDQVSAVDSYYYDDGCEPCGNDTFNREPFIVRFFSRFTEVREFAIVPLHAAPGDAVAEIDALYDVYLDVQEKWGLEDVMLMGDFNAGCSYVRPSQWSSIRLWTSPTFQWLIPDSADTTAKPTHCAYDRIVVAGMLLRGAVVPDSALPFNFQAAYGLSDQLAQAI SDHYPVEVMLK 10 RSLV-150 RNaseEPKSCDKTHTCPPCPAPELLGGSSVFLFPPKPKDTLMISR chain (FcB-NLGTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR linker-RNase) aminoEEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP acid sequenceASIEKTISKAKGQPREPQVYVLPPSRDELTKNQVSLLCLVKGFYPSDIAVEWESNGQPENNYLTWPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GKVDGASSPVNVSSPSVQDIKESRAKKFQRQHMDSDSS PSSSSTYCNQMMRRRNMTQGRCKPVNTFVHEPLVDVQNVCFQEKVTCKNGQGNCYKSNSSMHITDCRLTNGSRYPNCAYRTSPKERHIIVACEGSPYVPVHFDASVEDST 11 RSLV-151 DNaseDKTHTCPPCPAPELLGGSSVFLFPPKPKDTLMISRTPEVT chain (Fc-NLGCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY linker-DNase) aminoNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEK acid sequenceTISKAKGQPREPQVYVYPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFALVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKVD GASSPVNVSSPSVQDILKIAAFNIQTFGRTKMSNATLVS YIVQILSRYDIALVQEVRDSHLTAVGKLLDNLNQDAPDTYHYVVSEPLGRKSYKERYLFVYRPDQVSAVDSYYYDDGCEPCGNDTFNREPFIVRFFSRFTEVREFAIVPLHAAPGDAVAEIDALYDVYLDVQEKWGLEDVMLMGDFNAGCSYVRPSQWSSIRLWTSPTFQWLIPDSADTTAKPTHCAYDRIVVAGMLLRGAVVPDSALPFNFQAAYGLSDQLAQAISDH YPVEVMLK 12 RSLV-151 RNaseDKTHTCPPCPAPELLGGSSVFLFPPKPKDTLMISRTPEVT chain (Fc-NLGCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY linker-RNase) aminoNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEK acid sequenceTISKAKGQPREPQVYVLPPSRDELTKNQVSLLCLVKGFYPSDIAVEWESNGQPENNYLTWPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKVD GASSPVNVSSPSVQDIKESRAKKFQRQHMDSDSSPSSSS TYCNQMMRRRNMTQGRCKPVNTFVHEPLVDVQNVCFQEKVTCKNGQGNCYKSNSSMHITDCRLTNGSRYPNCAY RTSPKERHIIVACEGSPYVPVHFDASVEDST13 RSLV-152 (RNase- KESRAKKFQRQHMDSDSSPSSSSTYCNQMMRRRNMTQ Fc-NLG linker-GRCKPVNTFVHEPLVDVQNVCFQEKVTCKNGQGNCYK DNase) amino acidSNSSMHITDCRLTNGSRYPNCAYRTSPKERHIIVACEGSP sequenceYVPVHFDASVEDSTEPKSSDKTHTCPPCPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYVYPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFALVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKVDGASSPVNVSSPSVQDI LKIAAFNIQTFGRTKMSNATLVSYIVQILSRYDIALVQEVRDSHLTAVGKLLDNLNQDAPDTYHYVVSEPLGRKSYKERYLFVYRPDQVSAVDSYYYDDGCEPCGNDTFNREPFIVRFFSRFTEVREFAIVPLHAAPGDAVAEIDALYDVYLDVQEKWGLEDVMLMGDFNAGCSYVRPSQWSSIRLWTSPTFQWLIPDSADTTAKPTHCAYDRIVVAGMLLRGAVVPDSALPFNF QAAYGLSDQLAQAISDHYPVEVMLK 14RSLV-152 Fc DKTHTCPPCPAPELLGGSSVFLFPPKPKDTLMISRTPEVT domain chain aminoCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY acid sequenceNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYVLPPSRDELTKNQVSLLCLVKGFYPSDIAVEWESNGQPENNYLTWPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 15 RSLV-153 DNaseDKTHTCPPCPAPELLGGSSVFLFPPKPKDTLMISRTPEVT chain (Fc-NLGCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY linker-DNase) aminoNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEK acid sequenceTISKAKGQPREPQVYVYPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFALVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKVD GASSPVNVSSPSVQDILKIAAFNIQTFGRTKMSNATLVS YIVQILSRYDIALVQEVRDSHLTAVGKLLDNLNQDAPDTYHYVVSEPLGRKSYKERYLFVYRPDQVSAVDSYYYDDGCEPCGNDTFNREPFIVRFFSRFTEVREFAIVPLHAAPGDAVAEIDALYDVYLDVQEKWGLEDVMLMGDFNAGCSYVRPSQWSSIRLWTSPTFQWLIPDSADTTAKPTHCAYDRIVVAGMLLRGAVVPDSALPFNFQAAYGLSDQLAQAISDH YPVEVMLK 16 RSLV-153 RNaseKESRAKKFQRQHMDSDSSPSSSSTYCNQMMRRRNMTQ chain (RNase-Fc)GRCKPVNTFVHEPLVDVQNVCFQEKVTCKNGQGNCYK amino acid sequenceSNSSMHITDCRLTNGSRYPNCAYRTSPKERHIIVACEGSPYVPVHFDASVEDSTEPKSSDKTHTCPPCPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYVLPPSRDELTKNQVSLLCLVKGFYPSDIAVEWESNGQPENNYLTWPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK 17RSLV-154 (RNase- KESRAKKFQRQHMDSDSSPSSSSTYCNQMMRRRNMTQ Fc-NLG linker-GRCKPVNTFVHEPLVDVQNVCFQEKVTCKNGQGNCYK DNase) amino acidSNSSMHITDCRLTNGSRYPNCAYRTSPKERHIIVACEGSP sequenceYVPVHFDASVEDSTELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSRDELTKNQVNLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLNSTLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK VDGASSPVNVSSPSVQDILKIAAFNIQTFGRTKMSNATL VSYIVQILSRYDIALVQEVRDSHLTAVGKLLDNLNQDAPDTYHYVVSEPLGRKSYKERYLFVYRPDQVSAVDSYYYDDGCEPCGNDTFNREPFIVRFFSRFTEVREFAIVPLHAAPGDAVAEIDALYDVYLDVQEKWGLEDVMLMGDFNAGCSYVRPSQWSSIRLWTSPTFQWLIPDSADTTAKPTHCAYDRIVVAGMLLRGAVVPDSALPFNFQAAYGLSDQLAQAIS DHYPVEVMLK 18 Fc-NLG-linker-DKTHTCPPCPAPELLGGSSVFLFPPKPKDTLMISRTPEVT DNAse (controlCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY construct)NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKVD GASSPVNVSSPSVQDILKIAAFNIQTFGETKMSNATLVS YIVQILSRYDIALVQEVRDSHLTAVGKLLDNLNQDAPDTYHYVVSEPLGRNSYKERYLFVYRPDQVSAVDSYYYDDGCEPCRNDTFNREPFIVRFFSRFTEVREFAIVPLHAAPGDAVAEIDALYDVYLDVQEKWGLEDVMLMGDFNAGCSYVRPSQWSSIRLWTSPTFQWLIPDSADTTATPTHCAYDRIVVAGMLLRGAVVPDSALPFNFQAAYGLSDQLAQAISDHY PVEVMLK 19 DNASe-Fc (controlLKIAAFNIQTFGRTKMSNATLVSYIVQILSRYDIALVQEV construct)RDSHLTAVGKLLDNLNQDAPDTYHYVVSEPLGRKSYKERYLFVYRPDQVSAVDSYYYDDGCEPCGNDTFNREPFIVRFFSRFTEVREFAIVPLHAAPGDAVAEIDALYDVYLDVQEKWGLEDVMLMGDFNAGCSYVRPSQWSSIRLWTSPTFQWLIPDSADTTAKPTHCAYDRIVVAGMLLRGAVVPDSALPFNFQAAYGLSDQLAQAISDHYPVEVMLKEPKSSDKTHTCPPCPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK20 Mature wild type LKIAAFNIQTFGETKMSNATLVSYIVQILSRYDIALVQEVHuman DNase1 RDSHLTAVGKLLDNLNQDAPDTYHYVVSEPLGRNSYKERYLFVYRPDQVSAVDSYYYDDGCEPCGNDTFNREPAIVRFFSRFTEVREFAIVPLHAAPGDAVAEIDALYDVYLDVQEKWGLEDVMLMGDFNAGCSYVRPSQWSSIRLWTSPTFQWLIPDSADTTATPTHCAYDRIVVAGMLLRGAVVPDSAL PFNFQAAYGLSDQLAQAISDHYPVEVMLK 21Mature human LKIAAFNIQTFGETKMSNATLVSYIVQILSRYDIALVQEV DNase1 A114FRDSHLTAVGKLLDNLNQDAPDTYHYVVSEPLGRNSYKERYLFVYRPDQVSAVDSYYYDDGCEPCGNDTFNREP F IVRFFSRFTEVREFAIVPLHAAPGDAVAEIDALYDVYLDVQEKWGLEDVMLMGDFNAGCSYVRPSQWSSIRLWTSPTFQWLIPDSADTTATPTHCAYDRIVVAGMLLRGAVVPDSAL PFNFQAAYGLSDQLAQAISDHYPVEVMLK 22Mature human LKIAAFNIQTFGETKMSNATLVSYIVQILSRYDIALVQEV DNase1 G105RRDSHLTAVGKLLDNLNQDAPDTYHYVVSEPLGRNSYKERYLFVYRPDQVSAVDSYYYDDGCEPCRNDTFNREPAIVRFFSRFTEVREFAIVPLHAAPGDAVAEIDALYDVYLDVQEKWGLEDVMLMGDFNAGCSYVRPSQWSSIRLWTSPTFQWLIPDSADTTATPTHCAYDRIVVAGMLLRGAVVPDSAL PFNFQAAYGLSDQLAQAISDHYPVEVMLK 23Precursor human MRGMKLLGALLALAALLQGAVSLKIAAFNIQTFGETKM DNase1SNATLVSYIVQILSRYDIALVQEVRDSHLTAVGKLLDNLNQDAPDTYHYVVSEPLGRNSYKERYLFVYRPDQVSAVDSYYYDDGCEPCGNDTFNREPAIVRFFSRFTEVREFAIVPLHAAPGDAVAEIDALYDVYLDVQEKWGLEDVMLMGDFNAGCSYVRPSQWSSIRLWTSPTFQWLIPDSADTTATPTHCAYDRIVVAGMLLRGAVVPDSALPFNFQAAYGLSDQLAQ AISDHYPVEVMLK 24 Mature humanLKIAAFNIQTFGETKMSSATLVSYIVQILSRYDIALVQEV DNase1RDSHLTAVGKLLDNLNQDAPDTYHYVVSEPLGRNSYKE N18S/N106S/A114FRYLFVYRPDQVSAVDSYYYDDGCEPCGSDTFNREPFIVRFFSRFTEVREFAIVPLHAAPGDAVAEIDALYDVYLDVQEKWGLEDVMLMGDFNAGCSYVRPSQWSSIRLWTSPTFQWLIPDSADTTATPTHCAYDRIVVAGMLLRGAVVPDSALP FNFQAAYGLSDQLAQAISDHYPVEVMLK 25Mature human LKIAAFNIQTFG R TKMSNATLVSYIVQILSRYDIALVQEV DNase1RDSHLTAVGKLLDNLNQDAPDTYHYVVSEPLGR K SYKE E13R/N74K/A114F/RYLFVYRPDQVSAVDSYYYDDGCEPCGNDTFNREP F IV T205KRFFSRFTEVREFAIVPLHAAPGDAVAEIDALYDVYLDVQEKWGLEDVMLMGDFNAGCSYVRPSQWSSIRLWTSPTF QWLIPDSADTTA KPTHCAYDRIVVAGMLLRGAVVPDSA LPFNFQAAYGLSDQLAQAISDHYPVEVMLK 26 Mature humanLKIAAFNIQTFG R TKMS S ATLVSYIVQILSRYDIALVQEV DNase1RDSHLTAVGKLLDNLNQDAPDTYHYVVSEPLGR K SYKE E13R/N74K/A114F/RYLFVYRPDQVSAVDSYYYDDGCEPCG S DTFNREP F IV T205K/N18S/N106SRFFSRFTEVREFAIVPLHAAPGDAVAEIDALYDVYLDVQEKWGLEDVMLMGDFNAGCSYVRPSQWSSIRLWTSPTF QWLIPDSADTTA KPTHCAYDRIVVAGMLLRGAVVPDSA LPFNFQAAYGLSDQLAQAISDHYPVEVMLK 27 Mature humanKESRAKKFQRQHMDSDSSPSSSSTYCNQMMRRRNMTQ RNase1GRCKPVNTFVHEPLVDVQNVCFQEKVTCKNGQGNCYKSNSSMHITDCRLTNGSRYPNCAYRTSPKERHIIVACEGSP YVPVHFDASVEDST 28 Mature humanKESRAKKFQRQHMDSDSSPSSSSTYCNQMMRRRSMTQ RNase1GRCKPVNTFVHEPLVDVQNVCFQEKVTCKNGQGNCYK N34S/N76S/N88SSSSSMHITDCRLTSGSRYPNCAYRTSPKERHIIVACEGSPY VPVHFDASVEDST 29Precursor human MALEKSLVRLLLLVLILLVLGWVQPSLGKESRAKKFQR RNase1QHMDSDSSPSSSSTYCNQMMRRRNMTQGRCKPVNTFVHEPLVDVQNVCFQEKVTCKNGQGNCYKSNSSMHITDCRLTNGSRYPNCAYRTSPKERHIIVACEGSPYVPVHFDASV EDST 30 (Gly₄Ser)₃ linkerGGGGSGGGGSGGGGS 31 Gaussia luciferase MGVKVLFALICIAVAEA signal peptide32 Linker LEA(EAAAK)₄ALEA(EAAAK)₄ 33 O-linked CXXGG-T/S-C glycosylationconsensus 34 O-linked NST-E/D-A glycosylation consensus 35 O-linkedNITQS glycosylation consensus 36 O-linked QSTQS glycosylation consensus37 O-linked D/EFT-R/K-V glycosylation consensus 38 O-linked C-E/D-SNglycosylation consensus 39 O-linked GGSC-K/R glycosylation consensus 40VK3 light chain METPAQLLFLLLLWLPDTTG signal peptide 41 NLG linkerVDGASSPVNVSSPSVQDI 42 linker LEA(EAAAK)₄ALEA(EAAAK)₄ALE 43 Linker GGSG44 Linker GSAT 45 Human wild-typeEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR IgG1 Fc domainTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK 46 Mature humanMRICSFNVRSFGESKQEDKNAMDVIVKVIKRCDIILVME DNase1L3IKDSNNRICPILMEKLNRNSRRGITYNYVISSRLGRNTYKEQYAFLYKEKLVSVKRSYHYHDYQDGDADVFSREPFVVWFQSPHTAVKDFVIIPLHTTPETSVKEIDELVEVYTDVKHRWKAENFIFMGDFNAGCSYVPKKAWKNIRLRTDPRFVWLIGDQEDTTVKKSTNCAYDRIVLRGQEIVSSVVPKSNSVFDFQKAYKLTEEEALDVSDHFPVEFKLQSSRAFTNSKK SVTLRKKTKSKRS 47 Human Trex1MGPGARRQGRIVQGRPEMCFCPPPTPLPPLRILTLGTHTPTPCSSPGSAAGTYPTMGSQALPPGPMQTLIFFDMEATGLPFSQPKVTELCLLAVHRCALESPPTSQGPPPTVPPPPRVVDKLSLCVAPGKACSPAASEITGLSTAVLAAHGRQCFDDNLANLLLAFLRRQPQPWCLVAHNGDRYDFPLLQAELAMLGLTSALDGAFCVDSITALKALERASSPSEHGPRKSYSLGSIYTRLYGQSPPDSHTAEGDVLALLSICQWRPQALLRWVDAHARPFGTIRPMYGVTASARTKPRPSAVTTTAHLATTRNTSPSLGESRGTKDLPPVKDPGALSREGLLAPLGLLAILT LAVATLYGLSLATPGE 48Human DNase2 MIPLLLAALLCVPAGALTCYGDSGQPVDWFVVYKLPAL alphaRGSGEAAQRGLQYKYLDESSGGWRDGRALINSPEGAV (NP_001366.1)GRSLQPLYRSNTSQLAFLLYNDQPPQPSKAQDSSMRGHTKGVLLLDHDGGFWLVHSVPNFPPPASSAAYSWPHSACTYGQTLLCVSFPFAQFSKMGKQLTYTYPWVYNYQLEGIFAQEFPDLENVVKGHHVSQEPWNSSITLTSQAGAVFQSFAKFSKFGDDLYSGWLAAALGTNLQVQFWHKTVGILPSNCSDIWQVLNVNQIAFPGPAGPSFNSTEDHSKWCVSPKGPWTCVGDMNRNQGEEQRGGGTLCAQLPALWKAFQPL VKNYQPCNGMARKPSRAYKI 49human DNase2 beta MKQKMMARLLRTSFALLFLGLFGVLGAATISCRNEEGKAVDWFTFYKLPKRQNKESGETGLEYLYLDSTTRSWRKSEQLMNDTKSVLGRTLQQLYEAYASKSNNTAYLIYNDGVPKPVNYSRKYGHTKGLLLWNRVQGFWLIHSIPQFPPIPEEGYDYPPTGRRNGQSGICITFKYNQYEAIDSQLLVCNPNVYSCSIPATFHQELIHMPQLCTRASSSEIPGRLLTTLQSAQGQKFLHFAKSDSFLDDIFAAWMAQRLKTHLLTETWQRKRQELPSNCSLPYHVYNIKAIKLSRHSYFSSYQDHAKWCISQKGTKNRWTCIGDLNRSPHQAFRSGGFICTQNWQ IYQAFQGLVLYYESCK 50Fc region N83S EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYSSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK 51 Fc region with SCCEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK 52 Fc region with SSSEPKSSDKTHTSPPSPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K 53 LinkerLEA(EAAAK)₄ALEA(EAAAK)₄ALE 54 VK3LP leader METPAQLLFLLLLWLPDTTG 55Fc region with SCC, EPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTP238S, P331S PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE mutationsEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K 56 Fc region withEPKCSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISR P238S, P331STPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR mutationsEEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK 57 RSLV-327METPAQLLFLLLLWLPDTTGKESRAKKFQRQHMDSDSS (RNase-linker-HSA-PSSSSTYCNQMMRRRNMTQGRCKPVNTFVHEPLVDVQ linker-DNaseNVCFQEKVTCKNGQGNCYKSNSSMHITDCRLTNGSRYP E13R/N74K/A114F/NCAYRTSPKERHIIVACEGSPYVPVHFDASVEDSTGGGG T205K)SGGGGSGGGGSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGGGGSGGGGSGGGGSLKIAAFNIQTFGRTKMSNATLVSYIVQILSRYDIALVQEVRDSHLTAVGKLLDNLNQDAPDTYHYVVSEPLGRKSYKERYLFVYRPDQVSAVDSYYYDDGCEPCGNDTFNREPFIVRFFSRFTEVREFAIVPLHAAPGDAVAEIDALYDVYLDVQEKWGLEDVMLMGDFNAGCSYVRPSQWSSIRLWTSPTFQWLIPDSADTTAKPTHCAYDRIVVAGMLLRGAVVPDS ALPFNFQAAYGLSDQLAQAISDHYPVEVMLK

We claim:
 1. A heterodimer comprising a first nuclease domain, a secondnuclease domain, a first Fc domain and a second Fc domain, wherein thefirst nuclease domain is DNase1 and the second nuclease domain isRNase1, wherein the DNase1 is operably linked with or without a linkerto the N- or C-terminus of the first Fc domain, and the RNase1 isoperably linked with or without a linker to the N- or C-terminus of thesecond Fc domain.
 2. The heterodimer of claim 1, wherein the DNase 1 isoperably linked without a linker to the N-terminus of the first Fcdomain and the RNase1 is operably linked without a linker to theN-terminus of the second Fc domain.
 3. The heterodimer of claim 1,wherein the DNase 1 is operably linked with a linker to the N-terminusof the first Fc domain and the RNase1 is operably linked with a linkerto the N-terminus of the second Fc domain.
 4. The heterodimer of claim1, wherein the DNase 1 is operably linked with a linker to theN-terminus of the first Fc domain and the RNase1 is operably linkedwithout a linker to the C-terminus of the second Fc domain.
 5. Theheterodimer of claim 1, wherein the DNase 1 is operably linked without alinker to the N-terminus of the first Fc domain and the RNase1 isoperably linked without a linker to the C-terminus of the second Fcdomain.
 6. The heterodimer of claim 1, wherein the DNase 1 is operablylinked with a linker to the N-terminus of the first Fc domain and theRNase1 is operably linked with a linker to the C-terminus of the secondFc domain.
 7. The heterodimer of claim 1, wherein the DNase 1 isoperably linked with a linker to the C-terminus of the first Fc domainand the RNase1 is operably linked with a linker to the C-terminus of thesecond Fc domain.
 8. The heterodimer of claim 1, wherein the DNase 1 isoperably linked without a linker to the C-terminus of the first Fcdomain and the RNase1 is operably linked without a linker to theC-terminus of the second Fc domain.
 9. The heterodimer of claim 1,wherein the DNase 1 is operably linked with a linker to the C-terminusof the first Fc domain and the RNase1 is operably linked without alinker to the N-terminus of the second Fc domain.
 10. The heterodimer ofclaim 1, wherein the DNase 1 is operably linked with a linker to theC-terminus of the first Fc domain and the RNase1 is operably linked witha linker to the N-terminus of the second Fc domain.
 11. The heterodimerof any one of claims 1-10, wherein the RNase is a wild type humanRNase1, or a mutant RNase, such as an aglycosylated, underglycosylated,or deglycosylated RNase 1, such as human RNase1 N34S/N76S/N88S.
 12. Theheterodimer of claim 11, wherein the RNase is wild type human RNase1.13. The heterodimer of any one of claims 1-12, wherein the DNase is awild type human DNase1, or a mutant human DNase1 A114F, or anaglycosylated, underglycosylated, or deglycosylated mutant human DNase 1N18S/N106S/A114F.
 14. The heterodimer of any of the preceding claimswherein the first and second Fc domains comprise a hinge domain, a CH2domain and a CH3 domain.
 15. The heterodimer of claim 14, wherein thefirst and second Fc domains comprise a substitution of one or more ofthree hinge region cysteine residues with serine.
 16. The heterodimer ofclaim 15, wherein the first and second Fc domains comprise a mutationselected from the group consisting of SCC, SSS (residues 220, 226, and229), G236R, L328R, L234A, and L235A, numbering according to the EUindex.
 17. The heterodimer of claim 16, wherein the first and second Fcdomains comprise an SCC mutation (residues 220, 226, and 229), numberingaccording to the EU index.
 18. The heterodimer of any of the precedingclaims, wherein the first and second Fc domains comprise a P238S and aP331 mutation, numbering according to the EU index.
 19. The heterodimerof any of the preceding claims, wherein the first and second Fc domainscomprise one or more CH3 mutations to preferentially form heterodimers.20. The heterodimer of claim 19, wherein the first Fc domain comprisesCH3 mutations T350V, L351Y, F405A, and Y407V, and the second Fc domaincomprises CH3 mutations T350V, T366L, K392L, T394W, numbering accordingto the EU index.
 21. The heterodimer of any of the preceding claims,wherein the linker domain is a polypeptide linker, such as a gly-serlinker or an NLG linker (vdgasspvnvsspsvqdi).
 22. A heterodimercomprising a first and second polypeptide sequence selected from thegroup consisting of: (i) a first polypeptide comprising an amino acidsequence set forth in SEQ ID NO: 3, or a polypeptide comprising an aminoacid sequence at least 90% identical to the amino acid sequence setforth in SEQ ID NO:3; and a second polypeptide comprising an amino acidsequence set forth in SEQ ID NO: 4, or a polypeptide comprising an aminoacid sequence at least 90% identical to the amino acid sequence setforth in SEQ ID NO: 4, or (ii) a first polypeptide comprising an aminoacid sequence set forth in SEQ ID NO: 7, or a polypeptide comprising anamino acid sequence at least 90% identical to the amino acid sequenceset forth in SEQ ID NO:7; and a second polypeptide comprising an aminoacid sequence set forth in SEQ ID NO: 8, or a polypeptide comprising anamino acid sequence at least 90% identical to the amino acid sequenceset forth in SEQ ID NO:8, or (iii) a first polypeptide comprising anamino acid sequence set forth in SEQ ID NO:9, or a polypeptidecomprising an amino acid sequence at least 90% identical to the aminoacid sequence set forth in SEQ ID NO:9; and a second polypeptidecomprising an amino acid sequence set forth in SEQ ID NO: 10, or apolypeptide comprising an amino acid sequence at least 90% identical tothe amino acid sequence set forth in SEQ ID NO:10, or (iv) a firstpolypeptide comprising an amino acid sequence set forth in SEQ ID NO:11, or a polypeptide comprising an amino acid sequence at least 90%identical to the amino acid sequence set forth in SEQ ID NO: 11; and asecond polypeptide comprising an amino acid sequence set forth in SEQ IDNO: 12, or a polypeptide comprising an amino acid sequence at least 90%identical to the amino acid sequence set forth in SEQ ID NO:12, or (v) afirst polypeptide comprising an amino acid sequence set forth in SEQ IDNO: 15, or a polypeptide comprising an amino acid sequence at least 90%identical to the amino acid sequence set forth in SEQ ID NO: 15; and asecond polypeptide comprising an amino acid sequence set forth in SEQ IDNO:16, or a polypeptide comprising an amino acid sequence at least 90%identical to the amino acid sequence set forth in SEQ ID NO:16.
 23. Aheterodimer comprising a first nuclease domain, a second nuclease domainand a first Fc domain and a second Fc domain, wherein the first nucleasedomain is DNase1 and the second nuclease domain is RNase1, wherein (i)the DNase1 is operably linked with or without a linker to the N-terminusof the first Fc domain, and the RNase1 is operably linked with orwithout a linker to the C-terminus of the first Fc domain, or (i) theRNase1 is operably linked with or without a linker to the N-terminus ofthe first Fc domain, and the DNase1 is operably linked with or without alinker to the C-terminus of the first Fc domain.
 24. The heterodimer ofclaim 23, wherein the DNase 1 is operably linked without a linker to theN-terminus of the first Fc domain and the RNase1 is operably linked witha linker to the C-terminus of the first Fc domain.
 25. The heterodimerof claim 23, wherein the DNase 1 is operably linked with a linker to theN-terminus of the first Fc domain and the RNase1 is operably linked witha without a linker to the C-terminus of the first Fc domain.
 26. Theheterodimer of claim 23, wherein the RNase 1 is operably linked withouta linker to the N-terminus of the first Fc domain and the DNase 1 isoperably linked with a linker to the C-terminus of the first Fc domain.27. The heterodimer of claim 23, wherein the RNase 1 is operably linkedwith a linker to the N-terminus of the first Fc domain and the DNase 1is operably linked with a linker to the C-terminus of the first Fcdomain.
 28. The heterodimer of any one of claims 23-27, wherein theRNase is a wild type human RNase1, or a mutant RNase, such as anaglycosylated, underglycosylated, or deglycosylated RNase 1, such ashuman RNase1 N34S/N76S/N88S.
 29. The heterodimer of claim 28, whereinthe RNase is wild type human RNase1.
 30. The heterodimer of any one ofclaims 23-29, wherein the DNase is a wild type human DNase1, or a mutanthuman DNase1 A114F, or an aglycosylated, underglycosylated, ordeglycosylated mutant human DNase1 N18S/N106S/A114F.
 31. The heterodimerof any of the preceding claims wherein the first and second Fc domainscomprise a hinge domain, a CH2 domain and a CH3 domain.
 32. Theheterodimer of claim 31, wherein the first and second Fc domainscomprise a substitution of one or more of three hinge region cysteineresidues with serine.
 33. The heterodimer of claim 32, wherein the firstand second Fc domains comprise a mutation selected from the groupconsisting of SCC, SSS (residues 220, 226, and 229), G236R, L328R,L234A, and L235A, numbering according to the EU index.
 34. Theheterodimer of claim 33, wherein the first and second Fc domainscomprise an SCC mutation (residues 220, 226, and 229), numberingaccording to the EU index.
 35. The heterodimer of any of the precedingclaims, wherein the first and second Fc domains comprise a P238S and aP331 mutation, numbering according to the EU index.
 36. The heterodimerof any of the preceding claims, wherein the first and second Fc domainscomprise one or more CH3 mutations to preferentially form heterodimers.37. The heterodimer of claim 36, wherein the first Fc domain comprisesCH3 mutations T350V, L351Y, F405A, and Y407V, and the second Fc domaincomprises CH3 mutations T350V, T366L, K392L, T394W, numbering accordingto the EU index.
 38. The heterodimer of any of the preceding claims,wherein the linker domain is a polypeptide linker, such as a gly-serlinker or an NLG linker (vdgasspvnvsspsvqdi).
 39. A heterodimercomprising a first and second polypeptide sequence selected from thegroup consisting of: (i) a first polypeptide comprising an amino acidsequence set forth in SEQ ID NO:5, or a polypeptide comprising an aminoacid sequence at least 90% identical to the amino acid sequence setforth in SEQ ID NO:5; and a second polypeptide comprising an amino acidsequence set forth in SEQ ID NO:6, or a polypeptide comprising an aminoacid sequence at least 90% identical to the amino acid sequence setforth in SEQ ID NO:6, or (ii) a first polypeptide comprising an aminoacid sequence set forth in SEQ ID NO: 13, or a polypeptide comprising anamino acid sequence at least 90% identical to the amino acid sequenceset forth in SEQ ID NO: 13; and a second polypeptide comprising an aminoacid sequence set forth in SEQ ID NO: 14, or a polypeptide comprising anamino acid sequence at least 90% identical to the amino acid sequenceset forth in SEQ ID NO:14.
 40. A composition comprising the heterodimerof any of the preceding claims and a pharmaceutically acceptablecarrier.
 41. A nucleic acid molecule encoding the heterodimer of any ofthe preceding claims.
 42. A recombinant expression vector comprising anucleic acid molecule according to claim
 41. 43. A host cell transformedwith the recombinant expression vector according to claim
 42. 44. Amethod of making the heterodimer of any one of claims 1-39, comprising:providing a host cell comprising a nucleic acid sequence that encodesthe heterodimer; and maintaining the host cell under conditions in whichthe heterodimer is expressed.
 45. A method for treating or preventing acondition associated with an abnormal immune response, comprisingadministering to a subject an effective amount of a heterodimer of anyone of claims 1-39.
 46. The method of claim 45, wherein the condition isan autoimmune disease.
 47. The method of claim 46, wherein theautoimmune disease is selected from the group consisting ofinsulin-dependent diabetes mellitus, multiple sclerosis, experimentalautoimmune encephalomyelitis, rheumatoid arthritis, experimentalautoimmune arthritis, myasthenia gravis, thyroiditis, an experimentalform of uveoretinitis, Hashimoto's thyroiditis, primary myxoedema,thyrotoxicosis, pernicious anaemia, autoimmune atrophic gastritis,Addison's disease, premature menopause, male infertility, juvenilediabetes, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid,sympathetic ophthalmia, phacogenic uveitis, autoimmune haemolyticanaemia, idiopathic leucopenia, primary biliary cirrhosis, activechronic hepatitis Hbs-ve, cryptogenic cirrhosis, ulcerative colitis,Sjogren's syndrome, scleroderma, Wegener's granulomatosis, polymyositis,dermatomyositis, discoid LE, systemic lupus erythematosus (SLE), andconnective tissue disease.
 48. The method of claim 47, wherein theautoimmune disease is SLE.
 49. The method of claim 47, wherein theautoimmune disease is Sjogren's syndrome
 50. A method of treating SLEcomprising administering to a subject an amount of a heterodimereffective to degrade immune complexes containing RNA, DNA or both RNAand DNA, wherein the composition comprises a pharmaceutically acceptablecarrier and a heterodimer of any one of claims 1-39.
 51. A method oftreating Sjogren's syndrome comprising administering to a subject anamount of a heterodimer effective to degrade immune complexes containingRNA, DNA or both RNA and DNA, wherein the composition comprises apharmaceutically acceptable carrier and a heterodimer of any one ofclaims 1-39.